Complex Human Gut Microbiome Cultured In An Anaerobic Human Gut-On-A-Chip

ABSTRACT

A microfluidic device is directed to sustaining a complex microbial community in direct and indirect contact with living human intestinal cells in vitro. The device includes a first microchannel having cultured cells of a human intestinal epithelium and microbiota, the first microchannel further having a first level of oxygen. The device further includes a second microchannel having cultured cells of a vascular endothelium, the second microchannel further having a second level of oxygen. The device also includes a membrane located at an interface region between the first microchannel and the second microchannel, the membrane being composed of an oxygen-permeable material or further having pores via which oxygen flows between the first microchannel and the second microchannel to form a physiologically-relevant oxygen gradient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/722,658, filed Aug. 24, 2018, and U.S. Provisional PatentApplication No. 62/651,438, filed Apr. 2, 2018, each of which is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to supporting dynamicinteractions between living human intestinal epithelium and a directlyopposed complex community of living human aerobic and anaerobiccommensal gut microbes with a population diversity similar to thatobserved in a living human intestine.

BACKGROUND OF THE INVENTION

The diverse bacterial populations that comprise the commensal microbiotaof the human intestine play a central role in health and disease, yet nomethod is available to sustain these complex microbial communities indirect contact with living human intestinal cells in vitro. The presentdisclosure describes a human Gut-on-a-Chip (Gut Chip) microfluidicplatform that permits control and real-time assessment ofphysiologically-relevant oxygen gradients, and which enables co-cultureof living human intestinal epithelium in direct contact with stablecommunities of aerobic and anaerobic microbiota derived from human stoolspecimens. When compared to aerobic co-culture conditions, establishmentof a transluminal hypoxia gradient sustained higher microbial diversitywith over 200 unique operational taxonomic units (OTUs) from 11different genera, and an abundance of obligate anaerobic bacteria withratios of Firmicutes and Bacteroidetes similar to those observed inhuman feces, in addition to increasing intestinal barrier function. Theability to culture human intestinal epithelium overlaid by complex humangut microbial communities may enable investigations of host-microbiomeinteractions that were not possible previously, and serve as a discoverytool for development of new microbiome-related therapeutics, probiotics,and nutraceuticals.

One of the major recent paradigm shifts in medicine relates to therecognition of the central role that the microbiome composed ofhost-specific communities of commensal microbes plays in human healthand disease. Although human microbiota colonize mucosal surfaces ofvarious tissues, the gastrointestinal (GI) tract supports the greatestmass and diversity of microorganisms. Aerobic and anaerobic commensalgut microbiota are essential for maintenance of normal nutrientabsorption, drug metabolism, and immune responses, as well as forprotection against infectious pathogens. Conversely, changes orimbalances in the microbial community within the intestine cancontribute to development of a broad range of pathological disorderswithin and beyond the GI system, including inflammatory bowel disease,colorectal cancer, radiation enteropathy, diabetes, hepatic steatosis,obesity, and rheumatoid arthritis. Thus, the establishment andpreservation of balanced host-intestinal microbiome interactions are keyrequirements for maintaining gut homeostasis and human health.

Analysis of gut-microbiome crosstalk has almost exclusively relied ongenomic or metagenomic analysis of samples collected in vivo because nomethod exists to establish stable complex communities of gut commensalmicrobes in direct contact with intestinal epithelium in vitro. Althoughanimal models have been used to analyze host-microbiome interactions andtheir contributions to pathophysiology, microbiota differ betweendifferent species.

Existing in vitro models, such as Transwell inserts, have been used tostudy human host-microbe interactions; however, these studies can onlybe carried out over a period hours before bacterial overgrowth leads tocell injury and death. More advanced models, such as organoid cultures,have shown great promise for studying host-microbiome interactions, butthey are limited in providing a vascular interface and oxygen gradientswith below 1% luminal oxygen levels required for co-culture of certainstrict anaerobes. Human intestinal epithelial cells have been grown in amicrofluidic culture device separated by a nanoporous membrane from asingle facultative anaerobic bacterium (Lactobacillus rhamnosus GG) andan obligate anaerobe (Bacteroides caccae) cultured under anaerobicconditions in a parallel channel, which can permit analysis of theeffects of soluble mediators, but not the impact of direct contactbetween host cells and a complex community of commensal microbes. A2-channel, microfluidic, human Gut Chip device has been previouslydescribed as being lined by human Caco-2 intestinal epithelial cellsculture under dynamic fluid flow and peristalsis-like mechanicaldeformations, which enabled establishment of stable co-cultures of ahuman villus intestinal epithelium in direct contact with up to 8different strains of human commensal gut microbes for weeks in vitrounder oxygenated conditions¹, but the living intestinal microbiomecontains hundreds of different types of bacteria that are anaerobes aswell as aerobes.

Thus, there is a great need for experimental models that can sustaincomplex populations of human aerobic and anaerobic microbiota in contactwith living human tissues to analyze dynamic and physiologicallyrelevant human host-microbiome interactions. According to another need,an experimental system is required that can support dynamic interactionsbetween living human intestinal epithelium and a directly apposedcomplex community of living human aerobic and anaerobic commensal gutmicrobes with a population diversity similar to that observed in livinghuman intestine.

SUMMARY OF THE INVENTION

Embodiment A1. According to one embodiment of the present disclosure, amicrofluidic device is directed to sustaining a complex microbialcommunity in direct and indirect contact with living human intestinalcells in vitro. The device includes a first microchannel having culturedcells of a human intestinal epithelium and microbiota, the firstmicrochannel further having a first level of oxygen. The device furtherincludes a second microchannel having cultured cells of a vascularendothelium, the second microchannel further having a second level ofoxygen. The device also includes a membrane located at an interfaceregion between the first microchannel and the second microchannel, themembrane being composed of an oxygen-permeable material or furtherhaving pores via which oxygen flows between the first microchannel andthe second microchannel to form a physiologically-relevant oxygengradient.

Embodiment A2. The microfluidic device of embodiment A1, furthercomprising a plurality of microscale oxygen sensors embedded in thefirst microchannel and the second microchannel, the plurality ofmicroscale oxygen sensors providing real-time oxygen measurements basedon non-invasive monitoring of the physiologically-relevant oxygengradient.

Embodiment A3. The microfluidic device of embodiment A2, wherein theplurality of microscale oxygen sensors contain oxygen-quenchedfluorescent particles.

Embodiment A4. The microfluidic device of embodiment A3, wherein theoxygen-quenched fluorescent particles are suspended in apolydimethylsiloxane (PDMS) polymer or other gas-permeable polymer.

Embodiment A5. The microfluidic device of embodiment A3, wherein theoxygen-quenched fluorescent particles are cured in a film having athickness of between about and 1,000 micrometers (μm).

Embodiment A6. The microfluidic device of embodiment A3, wherein theoxygen-quenched fluorescent particles are in the form of discs having adiameter of about 0.1-5 millimeters (mm).

Embodiment A7. The microfluidic device of embodiment A2, wherein theplurality of microscale oxygen sensors are placed directly on aninterior surface of at least one of the first microchannel and thesecond microchannel.

Embodiment A8. The microfluidic device of embodiment A2, wherein theplurality of microscale oxygen sensors of are placed at an inlet region,a middle region, and an outlet region of each of the first microchanneland the second microchannel.

Embodiment A9. The microfluidic device of embodiment A2, wherein changesin fluorescent intensities of the plurality of microscale oxygen sensorsare caused by oxygen tension, the changes being indicative of oxygenconcentrations.

Embodiment A10. The microfluidic device of embodiment A1, wherein thefirst microchannel is a top microchannel and the second microchannel isa bottom microchannel.

Embodiment A11. The microfluidic device of embodiment A1, wherein thecultured cells of the vascular endothelium are human intestinalmicrovascular endothelia cells (HIMECs).

Embodiment A12. The microfluidic device of embodiment A1, wherein thephysiologically-relevant oxygen gradient is a hypoxia gradient.

Embodiment B1. According to another embodiment of the presentdisclosure, an in vitro system is directed to emulating a living humanintestine. The system includes a hypoxic chamber containing living humancommensal gut microbes and cultured cells of a human intestinalepithelium in which the microbes are in direct and indirect contact withthe cultured cells. The hypoxic chamber is configured to establish aphysiologically-relevant oxygen gradient across the layer of microbesand cultured cells.

Embodiment B2. The in vitro system of embodiment B1, further comprisinga plurality of microscale oxygen sensors providing real-time oxygenmeasurements based on non-invasive monitoring of the oxygen gradient.

Embodiment B3. The in vitro system of embodiment B1, wherein thecultured cells include one or more of mammalian cells, gut cells ofinsects, and gut cells of amphibians.

Embodiment B4. The in vitro system of embodiment B1, further comprisinga mucus layer in contact with the cultured cells, the mucus layer beingsecreted by the cultured cells or separately provided.

Embodiment B5. The in vitro system of embodiment B1, wherein themicrobes are contained in a layer.

Embodiment B6. The in vitro system of embodiment B5, wherein the layerof microbes and the cultured cells are placed within a microchannel.

Embodiment B7. The in vitro system of embodiment B6, wherein themicrochannel is a top microchannel that is separated from a bottommicrochannel via a membrane located at an interface region, the membranebeing oxygen permeable or having a plurality of pores via which oxygenflows between the top microchannel and the bottom microchannel toachieve the oxygen gradient.

Embodiment B8. The in vitro system of embodiment B7, further comprisinga plurality of microscale oxygen sensors providing real-time oxygenmeasurements based on non-invasive monitoring of the oxygen gradient,the plurality of microscale oxygen sensors being embedded in at leastone of the top microchannel and the bottom microchannel.

Embodiment B9. The in vitro system of embodiment B7, wherein the oxygengradient is based on top oxygen permeability through a device body to anexternal environment maintained at about 0 percent oxygen.

Embodiment B10. The in vitro system of embodiment B1, further comprisinga Charge-Coupled Device (CCD) camera, a photodiode, or otherlight-sensing modality via which fluorescence read-out measurementsprovide the real-time oxygen measurements in a non-invasive manner.

Embodiment C1. According to another embodiment of the presentdisclosure, a method is directed to establishing a stable complexcommunity of gut commensal microbes in vitro. The method includesproviding cultured cells of an intestinal epithelium and microbiota inan environment having a first level of oxygen, the microbiota being indirect and indirect contact with the intestinal epithelium. The methodalso includes providing cultured cells of a vascular endothelium in anenvironment having a second level of oxygen, the second level of oxygenhaving a greater oxygen concentration than the first level of oxygen.The method further includes facilitating the flux of oxygen between thefirst level of oxygen and the second level of oxygen to form aphysiologically-relevant oxygen gradient.

Embodiment C2. The method of embodiment C1, further comprisingmonitoring the oxygen gradient in a non-invasive manner, and measuringvalues of the oxygen gradient.

Embodiment C3. The method of embodiment C1, further comprising measuringthe values of the oxygen gradient via a non-invasive fluorescenceread-out.

Embodiment C4. The method of embodiment C1, further comprising providingoxygenation of the cultured cells of the intestinal epithelium and thecultured cells of the vascular endothelium while simultaneouslyproviding an anaerobic environment for growth of obligate anaerobes.

Embodiment C5. The method of embodiment C1, further comprising achievingan oxygen concentration of less than approximately 0.5-2.0% in the firstlevel of oxygen.

Embodiment C6. The method of embodiment C1, wherein the non-invasivemanner includes positioning a camera directly beneath the cultured cellsof the intestinal epithelium, the camera providing images of thecultured cells of the intestinal epithelium and microbiota.

Embodiment C7. The method of embodiment C1, wherein the cultured cellsinclude one or more cells of non-gut organs with low oxygen tension.

Embodiment C8. The method of embodiment C7, wherein the non-gut organsinclude one or more of an oral mucosa, urinary tract, and genitalmucosa.

Embodiment D1. According to yet another embodiment of the presentdisclosure, a microfluidic device has a first microchannel comprising aplurality of living parenchyma cells in direct contact with a pluralityof living microbes, wherein the microbes are derived from a mammalianfecal sample.

Embodiment D2. The microfluidic device of embodiment D1, wherein theparenchyma cells are selected from the group consisting of cells of thesmall intestine, ilea, duodenum, lung, alveolar, and skin.

Embodiment D3. The microfluidic device of embodiment D1, wherein themammal is a human.

Embodiment D4. The microfluidic device of embodiment D1, furthercomprising a second microchannel.

Embodiment D5. The microfluidic device of embodiment D4, wherein thefirst and second microchannels comprise media.

Embodiment D6. The microfluidic device of embodiment D4, wherein themedia in the second microchannel is oxygenated.

Embodiment D7. The microfluidic device of embodiment D4, wherein thedevice has a gas gradient.

Embodiment D8. The microfluidic device of embodiment D7, wherein the gasin the first microchannel is at a lower concentration than the gas inthe second microchannel.

Embodiment D9. The microfluidic device of embodiment D8, wherein the gasis selected from the group consisting of oxygen, nitrogen and carbondioxide.

Embodiment D10. The microfluidic device of embodiment D4, wherein thesecond microchannel comprises living endothelial cells.

Embodiment D11. The microfluidic device of embodiment D1, wherein theplurality of microbes comprises both anaerobic bacteria and aerobicbacteria.

Embodiment D12. The microfluidic device of embodiment D1, wherein theplurality of microbes comprises both Firmicutes phyla and Bacteroidetesphyla.

Embodiment D13. The microfluidic device of embodiment D1, wherein theFirmicutes species are selected from the group consisting ofAkkermansia, Osciliospira, Blautia and Suterella species.

Embodiment D14. The microfluidic device of embodiment D1, wherein theplurality of microbes comprises Coprococcus, Anaerobacillus,Bifidobacterium, and Peptoniphilus species.

Embodiment D15. The microfluidic device of embodiment D1, wherein theplurality of microbes comprises at least 8 different genera of bacteriafound in human feces.

Embodiment D16. The microfluidic device of embodiment D15, wherein theplurality of microbes comprises at least 11 different genera of bacteriafound in human feces.

Embodiment E1. According to yet another embodiment of the presentdisclosure, a method includes a) providing, i) a mammalian fecal samplecomprising living microbes, and ii) a solution of fluid; b) suspendingat least a portion of the fecal sample in the solution so as to create afecal slurry comprising living microbes; c) filtering the slurry so asto generate a microbiome stock derived directly from a fecal sample; d)diluting the microbiome stock so as to create a diluted stock; e)introducing the diluted stock into a microfluidic device; and f)culturing the diluted stock in the microfluidic device so as to create acultured microbiome of living microbes.

Embodiment E2. The method of embodiment E1, wherein one or more steps ofthe method take place in an anaerobic chamber.

Embodiment E3. The method of embodiment E2, wherein the suspending takesplace inside the anaerobic chamber.

Embodiment E4. The method of embodiment E1, wherein the mammalian fecalsample is from a human.

Embodiment E5. The method of embodiment E4, wherein the human isselected from the group consisting of a preterm infant, infant, child,teen, and an adult.

Embodiment E6. The method of embodiment E4, wherein the fecal sample isfrom a diaper.

Embodiment E7. The method of embodiment E4, wherein the fecal sample isa stool sample.

Embodiment E8. The method of embodiment E4, wherein the fecal sample wasobtained during a medical procedure.

Embodiment E9. The method of embodiment E1, wherein the fecal portion issuspended at 100 mg·ml⁻¹ for creating the fecal slurry.

Embodiment E10. The method of embodiment E4, wherein the fecal sample ofstep a) was not passed through another mammal.

Embodiment E10. The method of embodiment E4, wherein the fecal sample ofstep a) was not cultured in vitro.

Embodiment E11. The method of embodiment E4, wherein the fecal samplecomprises both anaerobic bacteria and aerobic bacteria.

Embodiment E12. The method of embodiment E1, wherein the diluting of themicrobiome stock generates a concentration of microbes of approximately1×10⁷ CFU ml⁻¹.

Embodiment E13. The method of embodiment E1, wherein the filtering ofstep c) is done with a filter that has a 40 μm pore size or less.

Embodiment E14. The method of embodiment E1, wherein the culturedmicrobiome comprises organisms from both the Firmicutes phyla and theBacteroidetes phyla.

Embodiment E15. The method of embodiment E1, wherein the culturedmicrobiome comprises species selected from the group consisting ofAkkermansia, Oscillospira, Blautia and Suterella species.

Embodiment E16. The method of embodiment E1, wherein the culturedmicrobiome comprises Coprococcus, Anaerobacillus, Bifidobacterium, andPeptoniphilus species.

Embodiment E17. The method of embodiment E1, wherein the culturedmicrobiome comprises at least 8 different genera of bacteria found inhuman feces.

Embodiment E18. The method of embodiment E1, wherein the culturedmicrobiome comprises at least 11 different genera of bacteria found inhuman feces.

Embodiment E19. The method of embodiment E13, further comprising g)flushing media through the cultured microbiome in the microfluidicdevice.

Embodiment E20. The method of embodiment E19, wherein the flushingprovides a sample of cultured living microbes.

Embodiment E21. The method of embodiment E1, wherein the microfluidicdevice comprises a first microchannel comprising a plurality of livingparenchyma cells.

Embodiment E22. The method of embodiment E21, wherein the introducing ofstep e) results in the parenchyma cells being in direct contact with aplurality of living microbes.

Embodiment E23. The method of embodiment E22, wherein the parenchymacells are selected from the group consisting of cells of the smallintestine, ilea, duodenum, lung, alveolar, and skin.

Embodiment E24. The method of embodiment E23, wherein the cells areintestinal epithelial cells.

Embodiment E25. The method of embodiment E21, wherein the microfluidicdevice further comprises a second microchannel.

Embodiment E26. The method of embodiment E25, wherein the first andsecond microchannels comprise media.

Embodiment E27. The method of embodiment E26, wherein the media in thesecond microchannel is oxygenated.

Embodiment E28. The method of embodiment E26, wherein the microfluidicdevice has a gas gradient.

Embodiment E29. The method of embodiment E28, wherein the gas in thefirst microchannel is at a lower concentration than the gas in thesecond microchannel.

Embodiment E30. The method of embodiment E29, wherein the gas isselected from the group consisting of oxygen, nitrogen and carbondioxide.

Embodiment E31. The method of embodiment E25, wherein the secondmicrochannel comprises living endothelial cells.

Embodiment E32. The method of embodiment E28, wherein the gas gradientprovides at least one hypoxic region in the first microchannel.

Embodiment E33. The method of embodiment E26, wherein the culturingcomprises flowing media at a flow rate.

Embodiment E34. The method of embodiment E26, wherein the secondmicrochannel is positioned below the first microchannel and separatedfrom the first microchannel by a membrane.

Embodiment E35. The method of embodiment E34, wherein oxygenated mediumflows through the second microchannel from external oxygenated mediumreservoirs.

Embodiment E36. The method of embodiment E35, wherein parenchyma cellsin the first microchannel get oxygen from the second microchannel.

Embodiment E37. The method of embodiment E1, wherein the culturing takesplace for at least 2 days.

Embodiment E38. The method of embodiment E1, wherein the culturing takesplace for at least 3 days.

Embodiment E39. The method of embodiment E1, wherein the culturing takesplace for at least 5 days.

Embodiment E40. The method of embodiment E38, wherein culturedmicrobiome comprises both anaerobic bacteria and aerobic bacteria.

Embodiment E41. The method of embodiment E40, wherein the culturedmicrobiome comprises at least 2 anaerobic species found in the fecalsample.

Embodiment E42. The method of embodiment E40, wherein the culturedmicrobiome comprises microbes from at least 2 genera found in the fecalsample.

Embodiment F1. According to yet another embodiment of the presentdisclosure, a method includes a) providing a microfluidic device and aportion of a mammalian fecal sample, the portion comprising livingmicrobes; b) introducing the portion into the microfluidic device; andc) culturing the living microbes in the microfluidic device so as tocreate a cultured microbiome.

Embodiment F2. The method of embodiment F1, wherein, prior to theintroducing of step b) the portion of the fecal sample is suspended in asterile solution so as to create a fecal slurry.

Embodiment F3. The method of embodiment F2, wherein the suspending takesplace inside an anaerobic chamber.

Embodiment F4. The method of embodiment F2, wherein, after thesuspending, the slurry is passed through a filter, so as to generate amicrobiome stock derived directly from a fecal sample.

Embodiment F5. The method of embodiment F4, wherein the filter has a 40μm pore size or less.

Embodiment F6. The method of embodiment F4, further comprising dilutingthe microbiome stock so as to create a diluted stock, the diluted stockbeing introduced in step b).

Embodiment F7. The method of embodiment F6, wherein the diluting themicrobiome stock generates a concentration of microbes of approximately1×10⁷ CFU ml⁻¹.

Embodiment F8. The method of embodiment F1, wherein the mammalian fecalsample is from a human.

Embodiment F9. The method of embodiment F8, wherein the human isselected from the group consisting of a preterm infant, infant, child,teen, and an adult.

Embodiment F10. The method of embodiment F8, wherein the fecal sample isfrom a diaper.

Embodiment F11. The method of embodiment F8, wherein the fecal sample isa stool sample.

Embodiment F12. The method of embodiment F8, wherein the fecal samplewas obtained during a medical procedure.

Embodiment F13. The method of embodiment F2, wherein the fecal portionis suspended at 100 mg·ml⁻¹.

Embodiment F14. The method of embodiment F8, wherein the fecal sample ofstep a) was not passed through another mammal.

Embodiment F15. The method of embodiment F8, wherein the fecal sample ofstep a) was not cultured in vitro.

Embodiment F16. The method of embodiment F1, wherein the fecal samplecomprises both anaerobic bacteria and aerobic bacteria.

Embodiment F17. The method of embodiment F16, wherein the culturedmicrobiome comprises at least one of the same anaerobic bacteria typesand aerobic bacteria types of the fecal sample.

Embodiment F18. The method of embodiment F1, wherein the culturedmicrobiome comprises both Firmicutes phyla and Bacteroidetes phyla.

Embodiment F19. The method of embodiment F1, wherein the culturedmicrobiome comprises species selected from the group consisting ofAkkermansia, Oscillospira, Blautia and Suterella species.

Embodiment F20. The method of embodiment F1, wherein the culturedmicrobiome comprises Coprococcus, Anaerobacillus, Bifidobacterium, andPeptoniphilus species.

Embodiment F21. The method of embodiment F1, wherein the culturedmicrobiome comprises at least 8 different genera of bacteria found inhuman feces.

Embodiment F22. The method of embodiment F1, wherein the culturedmicrobiome comprises at least 11 different genera of bacteria found inhuman feces.

Embodiment F23. The method of embodiment F1, further comprising d)flushing media through the cultured microbiome in the microfluidicdevice.

Embodiment F24. The method of embodiment F23, wherein the flushingprovides a sample of cultured living microbes.

Embodiment F25. The method of embodiment F1, wherein the microfluidicdevice comprises a first microchannel comprising a plurality of livingparenchyma cells.

Embodiment F26. The method of embodiment F25, wherein the introducing ofstep b) results in the parenchyma cells being in direct contact with aplurality of living microbes.

Embodiment F27. The method of embodiment F25, wherein the parenchymacells are selected from the group consisting of cells of the smallintestine, ilea, duodenum, lung, alveolar, and skin.

Embodiment F28. The method of embodiment F27, wherein the cells areintestinal epithelial cells.

Embodiment F29. The method of embodiment F25, wherein the microfluidicdevice further comprises a second microchannel.

Embodiment F30. The method of embodiment F29, wherein the first andsecond microchannels comprise media.

Embodiment F31. The method of embodiment F30, wherein the media in thesecond microchannel is oxygenated.

Embodiment F32. The method of embodiment F29, wherein the microfluidicdevice has a gas gradient.

Embodiment F33. The method of embodiment F32, wherein the gas in thefirst microchannel is at a lower concentration than the gas in thesecond microchannel.

Embodiment F34. The method of embodiment F33, wherein the gas isselected from the group consisting of oxygen, nitrogen and carbondioxide.

Embodiment F35. The method of embodiment F29, wherein the secondmicrochannel comprises living endothelial cells.

Embodiment F36. The method of embodiment F32, wherein the gas gradientprovides anaerobic conditions in the first microchannel.

Embodiment F37. The method of embodiment F25, wherein the introducing ofstep b) results in the parenchyma cells being in direct contact withliving obligate anaerobes.

Embodiment F38. The method of embodiment F1, wherein the culturingcomprises flowing media at a flow rate.

Embodiment F39. The method of embodiment F29, wherein the secondmicrochannel is positioned below the first microchannel and separatedfrom the first microchannel by a membrane.

Embodiment F40. The method of embodiment F39, wherein oxygenated mediumflows through the second microchannel from external oxygenated mediumreservoirs.

Embodiment F41. The method of embodiment F40, wherein parenchyma cellsin the first microchannel get oxygen from the second microchannel.

Embodiment F42. The method of embodiment F1, wherein the culturing takesplace for at least 2 days.

Embodiment F43. The method of embodiment F1, wherein the culturing takesplace for at least 3 days.

Embodiment F44. The method of embodiment F1, wherein the culturing takesplace for at least 5 days.

Embodiment F45. The method of embodiment F43, wherein culturedmicrobiome comprises both anaerobic bacteria and aerobic bacteria.

Embodiment F46. The method of embodiment F43, wherein the culturedmicrobiome comprises at least 2 anaerobic species found in the fecalsample.

Embodiment F47. The method of embodiment F43, wherein the culturedmicrobiome comprises microbes from at least 2 genera found in the fecalsample.

Embodiment G1. According to yet another embodiment of the presentdisclosure, a method includes a) providing a microfluidic device andliving microbes from the surface or contents of a body, orifice orcavity; b) introducing at least a portion of the living microbes intothe microfluidic device; and c) culturing the living microbes in themicrofluidic device so as to create a cultured microbiome.

Embodiment G2. The method of embodiment G1, where the surface of a bodyis skin.

Embodiment G3. The method of embodiment G1, wherein the content of abody is saliva.

Embodiment G4. The method of embodiment G1, wherein the body is the bodyof a mammal.

Embodiment G5. The method of embodiment G1, wherein the body is the bodyof a non-mammal.

Embodiment G6. The method of embodiment G5, wherein the non-mammal is abird.

Embodiment G7. The method of embodiment G1, wherein the culturingcomprises flowing media at a flow rate.

Embodiment G8. The method of embodiment G7, wherein the microfluidicdevice comprises a second microchannel positioned below a firstmicrochannel and separated from the first microchannel by a membrane.

Embodiment G9. The method of embodiment G8, wherein oxygenated mediumflows through the second microchannel from external oxygenated mediumreservoirs.

Embodiment G10. The method of embodiment G9, wherein living parenchymacells are in the first microchannel.

Embodiment G11. The method of embodiment G10, wherein the livingparenchyma cells get oxygen from the second microchannel.

Embodiment H1. According to yet another embodiment of the presentdisclosure, a method includes a) providing a microfluidic device, asource of microbes comprising living obligate anaerobes and livingparenchyma cells; and b) culturing the obligate anaerobes and theparenchyma cells in the microfluidic device such that at least a portionof the obligate anaerobes and at least a portion of the parenchyma cellsare in direct contact.

Embodiment H2. The method of embodiment H1, wherein the living obligateanaerobes are from the surface or contents of a body, orifice or cavity.

Embodiment H3. The method of embodiment H1, wherein the parenchyma cellsare human intestinal epithelial cells.

Embodiment H4. The method of embodiment H1, wherein, after theculturing, unknown microbes are identified.

Embodiment H5. The method of embodiment H1, wherein the culturingcomprises flowing media at a flow rate.

Embodiment H6. The method of embodiment H1, wherein the microfluidicdevice comprises a second microchannel positioned below a firstmicrochannel and separated from the first microchannel by a membrane.

Embodiment H7. The method of embodiment H6, wherein oxygenated mediumflows through the second microchannel from external oxygenated mediumreservoirs.

Embodiment H8. The method of embodiment H7, wherein parenchyma cells inthe first microchannel get oxygen from the second microchannel.

Embodiment H9. The method of embodiment H1, wherein the culturing takesplace for at least 2 days.

Embodiment H10. The method of embodiment H1, wherein the culturing takesplace for at least 3 days.

Embodiment H11. The method of embodiment H1, wherein the culturing takesplace for at least 5 days.

Embodiment H12. The method of embodiment H10, wherein culturedmicrobiome comprises both anaerobic bacteria and aerobic bacteria.

Embodiment H13. The method of embodiment H10, wherein the culturedmicrobiome comprises at least 2 anaerobic species found in the fecalsample.

Embodiment H14. The method of embodiment H10, wherein the culturedmicrobiome comprises microbes from at least 2 genera found in the fecalsample.

Embodiment I1. According to yet another embodiment of the presentdisclosure, a method includes a) providing, i) a liquid sample derivedfrom a culture of a plurality of microbes of different types, and ii) afirst microfluidic device capable of undergoing fluid flow, comprisingliving parenchymal cells in a first microchannel; and b) flowing saidliquid sample into said first microchannel so that at least a portion ofsaid sample contacts said living parenchymal cells.

Embodiment I2. The method of embodiment I1, further comprising c)detecting an effect of said liquid sample on said living parenchymalcells.

Embodiment I3. The method embodiment I1, wherein said parenchymal cellsare intestinal epithelial cells.

Embodiment I4. The method of embodiment I1, wherein said liquid sampleis derived from a second microfluidic device comprising a microbiome.

Embodiment I5. The method of embodiment 14, wherein said microbiome wascreated by inoculating said second microfluidic device with a pluralityof microbes derived from a fecal sample.

Embodiment I6. The method of embodiment 14, wherein said secondmicrofluidic device has an outlet and said liquid sample was collectedfrom said outlet as an effluent.

Embodiment I7. The method of embodiment 14, wherein said microbiomecomprises anaerobic and aerobic bacteria.

Embodiment I8. The method of embodiment 17, wherein said microbiome wasinoculated with a plurality of enterohemorrhagic Escherichia coli(EHEC).

Embodiment I9. The method of embodiment I1, wherein said firstmicrofluidic device was inoculated with a plurality of enterohemorrhagicEscherichia coli (EHEC).

Embodiment I10. The method of embodiment I1, wherein said liquid samplecomprises one or more metabolite compounds generated by said microbes.

Embodiment I11. The method of embodiment I1, wherein said liquid sampledoes not contain a living microbe.

Embodiment I12. The method of embodiment 13, wherein said intestinalepithelial cells are derived from a patent biopsy.

Embodiment I13. The method of claim 3, wherein said intestinalepithelial cells have a plurality of microvilli.

Embodiment I14. The method of embodiment I1, further comprising: i)providing a test compound, and ii) flowing said test substance into saidfirst microchannel.

Embodiment I15. The method of embodiment I1, wherein said firstmicrofluidic device further comprises a second microfluidic channel,separated by a membrane from said first microfluidic channel.

Embodiment I16. The method of embodiment 115, wherein said secondmicrofluidic channel comprises endothelial cells.

Embodiment I17. The method of embodiment 116, further comprising: i)providing a test compound, and ii) flowing said test substance into saidsecond microchannel.

Embodiment I18. The method of embodiment I10, wherein said one or moremetabolites are selected from the group consisting of 4-methyl benzoicacid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid.

Embodiment J1. According to yet another embodiment of the presentdisclosure, a method includes a) providing a microfluidic device capableof undergoing fluid flow, comprising living parenchymal cells in contactwith a plurality of diverse microbes in a first microchannel or chamber,wherein said microfluidic device has an outlet at the end of said firstmicrochannel or chamber; b) flowing liquid into said first microchannelor chamber; and c) collecting effluent at said outlet.

Embodiment J2. The method of embodiment J1, further comprising d)testing said effluent.

Embodiment J3. The method of embodiment J2, wherein said testingcomprises flowing at least a portion of said effluent into a secondmicrofluidic device comprising cells.

Additional aspects of the disclosure will be apparent to those ofordinary skill in the art in view of the detailed description of variousembodiments, which is made with reference to the drawings, a briefdescription of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation showing the position of a humanintestinal epithelium and microbiota on top.

FIG. 1B is a schematic representation of a Gut Chip with 6 oxygenquenched fluorescent particles embed in inlet, middle and outlet of topand bottom channels (T, top channel; B, bottom channel).

FIG. 1C is a graph showing sensitivity analysis of oxygen spots locatedin the Gut Chip in response to defined, standard oxygen concentrations.

FIG. 1D is a graph showing hypoxic chamber validation at various N₂inflow pressures.

FIG. 1E shows microscopic views of villus morphology of the human Caco-2intestinal epithelium (bar, 100 μm) and vascular endothelium (bottomleft; bar, 100 μm).

FIG. 1F is a graph showing oxygen concentration profiles withinaerobically- and anaerobically-cultured Gut Chips.

FIG. 2A is a graph showing oxygen concentration profiles in aerobic andanaerobic Gut Chips co-cultured with Bacteroides fragilis.

FIG. 2B shows representative vertical cross-sectional, confocalmicrographic views through the intestinal epithelium-microbiomeinterface within the Gut Chip.

FIG. 2C is a graph showing changes in apparent paracellular permeability(P_(app)).

FIG. 2D is a graph showing CFU counts of Bacteroides fragilisco-cultured in Gut Chip under aerobic and anaerobic conditions (n=3;*P<0.05, ***P<0.001).

FIG. 2E is a representative image confirming that Bacteroides fragilisresides on top of a mucus layer.

FIG. 2F shows representative images illustrating a continuous and densemucus blanket after a number of culture days.

FIG. 3A is a graph showing observed alpha diversity in microbiomesamples.

FIG. 3B is a graph showing changes in apparent paracellular permeability(Papp).

FIG. 3C is a graph showing aerobic, anaerobic, and human stool data.

FIG. 4A is a graph showing genera growing or maintained in the anaerobicsystem over time.

FIG. 4B is a graph showing a difference in abundance of bacteria inaerobic or anaerobic) when compared to a liquid culture, comparinggrowth at 3 days.

FIG. 4C is a graph showing a differential abundance in quantified generaacross 3 days of co-culture.

FIG. 5A is a representative optical image of an oxygen-sensing Gut Chip.

FIG. 5B is an image of the Gut Chip oxygen distribution in aerobic andanaerobic culture conditions.

FIG. 5C is a graph showing an accuracy analysis of oxygen spots locatedin the Gut Chip in response to defined, standard oxygen concentrations.

FIG. 5D is a graph representative of before and after plasma treatment.

FIG. 5E is a graph showing an altered thickness (150 μm vs. μ300 m) ofthe spot.

FIG. 5F shows representative images of the oxygen distribution fromaerobic to anaerobic conditions.

FIG. 6A is a schematic representation of a hypoxic chamber.

FIG. 6B is an image of the hypoxic chamber of FIG. 6A in use.

FIG. 7A is a graph showing effects on anaerobic culture of intestinalepithelium and vascular endothelium.

FIG. 7B is a graph showing changes in apparent paracellular permeability(Papp).

FIG. 8A shows representative images of immunofluorescence staining ofnuclei.

FIG. 8B is a graph showing the quantification of the percentage ofepithelial and endothelial cells that expressed HIF1-α (HIF1-α⁺ cells)after exposure to the conditions shown in a (n=3; *P<0.05, **P<0.01).

FIG. 9A shows a fragilis labeled with HADA.

FIG. 9B shows representative immunofluorescence micrographs of HADAlabeled Bacteroides fragilis.

FIG. 10A is a graph showing Caco-2 viability.

FIG. 10B is a graph showing changes in relative abundance of quantifiedmicrobial genera.

FIG. 10C is a graph showing genera abundance in a huma microbiome stock.

FIG. 10D is a graph showing a comparison between identified genera andpublicly available data.

FIG. 11 is a graph showing Genera growing or maintained in the anaerobicchip over time.

FIG. 12 is a table showing media tested for microbial diversity.

FIG. 13 is a perspective view of a bioreactor with an oxygen gradientconfiguration.

FIG. 14 is a longitudinal cross-sectional view along cross-sectionallines “14-14” of the bioreactor of FIG. 13 .

FIG. 15 is a lateral cross-sectional view along cross-sectional lines“15-15” of the bioreactor of FIG. 13 .

FIG. 16A shows a differential interference contract (DIC) microscopicimage of primary human ileum chips.

FIG. 16B shows a confocal fluorescence microscopic image of primaryhuman ileum chips.

FIG. 16C is a graph showing a co-culture stably maintained for up to atleast five days on-chip.

FIG. 16D is a table showing observed richness of various ileum samples.

FIG. 17A shows confocal fluorescence microscopic images with villusmorphology of a primary ileal epithelium stained for villin, F-actin andDAPI.

FIG. 17B shows an image of secreted mucus with alcian blue staining.

FIG. 17C is a graph with quantitation of alcian blue staining incultures shown in FIG. 17B.

FIG. 18A shows images of differential interference contrast of colonicepithelium.

FIG. 18B shows images of an entire colon epithelium.

FIG. 18C shows graphs representative of quantification of epitheliallesion areas.

FIG. 18D shows graphs representative of changes in levels of variousindicated cytokines released into a vascular channel of colon chips.

FIG. 19A shows an image of a heat-map of differentially expressed genes.

FIG. 19B shows a representative image of a gene enrichment analysis

FIG. 19C shows an image of a heat-map of chemotaxis and flagellarassembly pathways.

FIG. 19D shows a schematic illustrating key genes critical in regulatingchemotaxis and flagellar assembly in EHEC.

FIG. 19E shows plot images illustrating EHEC swimming motility tracking.

FIG. 19F shows a graph illustrating quantification of a fraction ofmoving EHEC.

FIG. 19G shows a graph illustrating mean velocity of each trackedbacterium.

FIG. 19H shows a graph illustrating a distance traveled by a movingbacteria.

FIG. 19I shows a graph illustrating Fli-C-luciferase expression levels.

FIG. 20A shows a Venn-diagram illustrating metabolomics analysisworkflow.

FIG. 20B shows a heat-map with 426 compounds produced by commensalbacteria.

FIG. 20C shows a plot of relative abundance for 30 microbiomemetabolites that were tested.

FIG. 20D shows a plot with results for FliC-luciferase (FliC-lux)screening for the selected metabolites.

FIG. 21A shows representative DIC images of a colon epithelium undervarious experimental conditions.

FIG. 21B shows images of an entire epithelial layer in a colon chipunder the same conditions.

FIG. 21C shows a plot representing quantification of an epithelial areasized under conditions shown in FIG. 21B.

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the invention is not intended to belimited to the particular forms disclosed. Rather, the invention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

As used herein, the phrases “linked,” “connected to,” “coupled to,” “incontact with” and “in communication with” refer to any form ofinteraction between two or more entities, including mechanical,electrical, magnetic, electromagnetic, fluidic, and thermal interaction.For example, in one embodiment, channels in a microfluidic device are influidic communication with cells and (optionally) a fluid reservoir (orother components). Two components may be coupled to each other eventhough they are not in direct contact with each other. For example, twocomponents may be coupled to each other through an intermediatecomponent (e.g. tubing or other conduit).

“Channels” are pathways (whether straight, curved, single, multiple, ina network, etc.) through a medium (e.g., silicon, plastic, etc.) thatallow for movement of liquids and gasses. Channels thus can connectother components, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, liquid-intake ports and gas vents.

“Microchannels” are channels with dimensions less than 1 millimeter andgreater than 1 micron. Additionally, the term “microfluidic” as usedherein relates to components where moving fluid is constrained in ordirected through one or more channels wherein one or more dimensions are1 mm or smaller (microscale). Microfluidic channels may be larger thanmicroscale in one or more directions, though the channel(s) will be onthe microscale in at least one direction. In some instances the geometryof a microfluidic channel may be configured to control the fluid flowrate through the channel (e.g. increase channel height to reduce shear).Microfluidic channels can be formed of various geometries to facilitatea wide range of flow rates through the channels.

The present invention contemplates a variety of “microfluidic devices,”including but not limited to microfluidic chips (such as that shown inFIGS. 1A and 1B). Some microfluidic devices comprise one or moremicrochannels with cells and culture media. For example, in oneembodiment, the present invention contemplates oxygenated medium flowingthrough the lower endothelium-lined vascular channel from externaloxygenated medium reservoirs. In this embodiment, epithelial cells inthe upper channel get oxygen from the lower channel (e.g. through aporous membrane, gel, pillars etc. or a combination thereof).

A “hypoxic chip” or “hypoxic microfluidic device” comprises a devicewith one or more hypoxic regions. Such regions have low levels ofoxygen, i.e. 5% or lower, more preferably 4% or lower, 3% or lower, 2%or lower, 1% or lower, 0.5% or lower, or 0.1% or lower. That is to say,the entire device need not be hypoxic. Moreover, it is not intended thatthe present invention be limited to how a hypoxic region is generated.Hypoxic conditions can be generated with a chamber (as shown in FIG. 6 )or without a chamber. Hypoxic conditions can be generated in amicrofluidic device that is not gas permeable, or that has a region thatis not gas permeable. Hypoxic conditions can be generated usingdeoxygenated media. Of course, these different approaches can becombined, if desired.

An “aerobic chip” is a microfluidic device where steps have not beentaken to create hypoxic conditions (e.g. no hypoxic chamber, nodeoxygenated media, etc.). Nonetheless, system components in an aerobicchip may regulate oxygen to support co-culture of anaerobes withmammalian cells. In particular, and without being bound by theory, themammalian cells consume oxygen that is predominantly delivered to themfrom their basal side; this reduces the concentration of oxygen on theanaerobes. In addition, and without being bound by theory, otherelements of the complex microbiome, for example aerobes present, alsoconsume remaining oxygen that may otherwise poison or inhibit growth ofthe anaerobes.

While a microbiome is exemplified herein using a fecal sample, thepresent invention contemplates other sources for generating a microbiomein a microfluidic device, including but not limited to skin, saliva,lung, armpit, toes, feet, etc. (e.g. any surface or contents of a body,orifice or cavity). Moreover, sources from both mammals and non-mammalscan be used.

According to the present disclosure, an experimental system has beendeveloped that can support dynamic interactions between living humanintestinal epithelium and a directly apposed complex community of livinghuman aerobic and anaerobic commensal gut microbes with a populationdiversity similar to that observed in living human intestine. To meetthis challenge, a human Gut Chip was modified by culturing humanintestinal microvascular endothelial cells (HIMECs) in a lower channel,integrating microscale oxygen sensors into the device for in situ oxygenmeasurements, and placing the Gut Chip within an engineered hypoxicchamber to establish a physiologically relevant oxygen gradient acrossthe Gut Chip vascular and epithelium channels. To emulate thephysiological human intestinal gut-microbiota interface on-chip, complexmicrobiota was derived from healthy human stool specimens, which havebeen maintained stably in gnotobiotic mice for multiple years. Thedisclosure below describes how to establish a hypoxia gradient acrossengineered tissue-tissue (endothelium-epithelium) interface of the GutChip, which allows stably co-culturing of complex communities ofanaerobic and aerobic human commensal gut bacteria in direct contactwith human villus intestinal epithelium while simultaneously monitoringoxygen levels for multiple days in vitro.

Referring to FIGS. 1A-1F, schematics and data illustrate anoxygen-sensitive human Gut chip microfluidic device. FIG. 1A a schematicrepresentation showing the position of a human intestinal epithelium andmicrobiota on top and further shows a vascular endothelium on a bottomside of the matrix-coated porous membrane within a 2-channelmicrofluidic device in presence of oxygen gradients. High and low levelsof oxygen concentration are also illustrated, with high levels beinggenerally towards the bottom and high levels being generally towards thetop. By way of example,

Further referring to FIG. 1A, and by way of example, a microfluidicdevice 100 is configured to sustain a complex microbial community indirect and indirect contact with living human intestinal cells in vitro.The microfluidic device 100 includes a first microchannel 102 that haswithin cultured cells 104 of a human intestinal epithelium andmicrobiota. The first microchannel 102 has a first level of oxygen 108.The microfluidic device 100 further includes a second microchannel 110that has within cultured cells 112 of a vascular endothelium. The secondmicrochannel 110 has a second level of oxygen 114 that has a greateroxygen concentration than the first level of oxygen 108. In thisexample, the first microchannel 102 is a top microchannel and the secondmicrochannel 110 is a bottom microchannel.

The microfluidic device 100 further includes a membrane 116 that islocated at an interface region between the first microchannel 102 andthe second microchannel 110. The membrane 116 has a first surface 118facing the first microchannel 102 and a second surface 120 facing thesecond microchannel 110. The membrane is composed of an oxygen-permeablematerial or has a plurality of pores via which oxygen flows between thefirst microchannel 102 and the second microchannel 110 to form aphysiologically-relevant oxygen gradient across the first microchannel102 and the second microchannel 110.

The microfluidic device 100 optionally includes a plurality ofmicroscale oxygen sensors 122 that contain oxygen-quenched fluorescentparticles. The plurality of microscale oxygen sensors 122 are optionallyplaced directly on an interior surface of at least one of the firstmicrochannel 102 and the second microchannel 110. The plurality ofmicroscale oxygen sensors 122 are optionally placed at an inlet region124, a middle region 126, and an outlet region 128 of each of the firstmicrochannel 102 and the second microchannel 110. The oxygen-quenchedfluorescent particles are optionally suspended in a polydimethylsiloxane(PDMS) polymer or other gas-permeable polymer. Optionally yet, theoxygen-quenched fluorescent particles are cured in a film having athickness of between about 50 and 1,000 micrometers (μm). In anotheralternative embodiment, the oxygen-quenched fluorescent particles are inthe form of discs having a diameter of about 0.1-5 millimeters (mm).Optionally yet, changes in fluorescent intensities of the plurality ofmicroscale oxygen sensors 122 are caused by oxygen tension, the changesbeing indicative of oxygen concentrations. Other features orconfigurations of the microfluidic device 100 are described below inaccordance with applicable experimental studies and data.

FIG. 1B shows a Gut Chip with 6 oxygen quenched fluorescent particlesembed in inlet, middle and outlet of top and bottom channels (T, topchannel; B, bottom channel). FIG. 1C shows sensitivity analysis ofoxygen spots located in the Gut Chip in response to defined, standardoxygen concentrations. FIG. 1D hypoxic chamber validation at various N₂inflow pressures and further shows N₂ introduced into the chamber at 81mL min⁻¹, 162 mL min⁻¹, or 243 mL min⁻¹ for 1 h when gas flow wasstopped and chamber was allowed to recover (n=3, shaded regions arestandard deviation). FIG. 1E shows villus morphology of the human Caco-2intestinal epithelium (bar, 100 μm) and vascular endothelium (bottomleft; bar, 100 μm), and further shows the human Caco-2 intestinalepithelium and vascular endothelium cultured for 6 days in the Gut Chipunder anaerobic condition, when viewed from above by DIC and phasecontrast imaging, respectively, or by immunofluorescence staining forthe tight junction protein, ZO-1 (red, top right; bar, 100 μm) andendothelial cell junction-associated protein, VE-cadherin (red, bottomright; bar, 20 μm). Gray indicates DAPI-stained nuclei. White dashedlines indicate borders of oxygen sensor spots). FIG. 1F shows oxygenconcentration profiles within aerobically- and anaerobically-culturedGut Chips, and further shows representative pseudocolor insets thatindicate average oxygen concentration in aerobic chip (1), and inlet(2), middle (3) and outlet (4) of anaerobically-cultured epitheliumchannel at day 7 of culture.

Referring to FIGS. 2A-2F, representative images and data show co-cultureof human intestinal epithelium and obligate anaerobe, Bacteroidesfragilis, on-chip. FIG. 2A shows oxygen concentration profiles inaerobic and anaerobic Gut Chips co-cultured with Bacteroides fragilis.FIG. 2B shows vertical cross-sectional, confocal micrographic viewsthrough the intestinal epithelium-microbiome interface within the GutChip, and further shows the Gut Chip cultured under anaerobic condition,when immunostained for villin, ZO-1 nuclei with DAPI (bar, 50 μm). B.fragilis is HADA labeled. FIG. 2C shows changes in apparent paracellularpermeability (P_(app)), which is measured by quantitating cascade bluetransport across the tissue-tissue interface within the Gut Chipmicrodevices co-cultured with Bacteroides fragilis under aerobic andanaerobic conditions (n=4; * P<0.05). FIG. 2D shows CFU counts ofBacteroides fragilis co-cultured in Gut Chip under aerobic and anaerobicconditions (n=3; * P<0.05, *** P<0.001). FIG. 2E shows cross-sectionalfluorescence microscopic view of the Caco2 epithelium (nuclei stained inblue with DAPI), overlying mucus layer stained with Alexa Fluor488-conjugated WGA (yellow), and B. fragilis bacteria (GalCCP labelled,white) when co-cultured in the intestine chip (scale bar, 10 μm). FIG.2F shows SEM views of the apical surface of the Caco2 epithelium in theintestine chip comparing the morphology on day 4 of culture before itaccumulates a mucus layer and when the surface microvilli are visible(top) versus when Bacteroides fragilis have been added on day 12 afterthe mucus layer has accumulated, which can be seen as a dense mat thatseparates the bacteria from the epithelial cell surface (bottom) (scalebar, 2 μm).

Referring to FIGS. 3A-3C, representative graphs are generally directedto the analysis of the diversity and relative abundance of microbiotaco-cultured in gut Chips under aerobic and anaerobic conditions. FIG. 3Ashows observed alpha diversity in microbiome samples in both anaerobicand aerobic conditions, across 3 days of co-culturing of a microbiomesample with human intestinal epithelium. FIG. 3B shows changes inapparent paracellular permeability (Papp) measured by quantitatingcascade blue transport across the tissue-tissue interface within the GutChip microdevices after diverse microbiome co-culture, under aerobic andanaerobic conditions (n=4; *P<0.05, ***P<0.001). FIG. 3C shows aerobic,anaerobic, and human stool data.

Referring to FIGS. 4A-4C, representative graphs are generally directedto showing hypoxic Gut Chip-microbiome co-culture that enhances thegrowth of multiple genera compared to conventional liquid culture oraerobic chip system. FIG. 4A shows genera growing or maintained in theanaerobic system over time. FIG. 4B shows a difference in abundance ofbacteria in aerobic or anaerobic) when compared to a liquid culture,comparing growth at 3 days. In FIG. 4C, which shows a differentialabundance in quantified genera across 3 days of co-culture, thedifferential abundance was determined using DESeq2 comparing theanaerobic read counts with the aerobic ones (as disclosed in the methodsof the present disclosure).

Referring to FIGS. 5A-5F, images and graphs represent an oxygen-sensingGut Chip 200. FIG. 5A shows the oxygen-sensing Gut Chip 200, and FIG. 5Bshows the Gut Chip oxygen distribution in aerobic and anaerobic cultureconditions. FIG. 5C shows an accuracy analysis of oxygen spots locatedin the Gut Chip 200 in response to defined, standard oxygenconcentrations. FIG. 5D shows before and after plasma treatment of theGut Chip 200. FIG. shows an altered thickness (150 μm vs. μ300 m) of thespot. FIG. 5F shows the oxygen distribution from aerobic to anaerobicconditions.

Referring to FIGS. 6A and 6B, representative images show a hypoxicchamber 300. In FIG. 6A, which is a schematic representation of ahypoxic chamber 300, a left image shows an exploded view of the hypoxicchamber 300, a middle image shows a linear positioning system 302 forindexed motions of the camera to any sensor spot along the chip orbetween the chips, and a right image shows rendering of a hypoxic farm304 on imaging stand for monitoring of sensors without removing chipsfrom hypoxic chamber 300. In FIG. 6B, which is an image of the hypoxicchamber 300 of FIG. 6A in use, chips 306 are placed in a hypoxic region308 of the chamber 300 with media for the epithelium channel 310(exposed to oxygen). Media reservoirs for the vascular channels 312(inside the anaerobic chamber) are maintained at normoxia. The chamber300 is purged with N₂ flow 314 through a bubbler 316.

Referring to FIGS. 7A and 7B, graphs show effects on anaerobic cultureand changes in apparent paracellular permeability. In FIG. 7A, effectson anaerobic culture of intestinal epithelium and vascular endotheliumare assessed by quantifying LDH release from cells (data are presentedas fold change in LDH levels relative to the aerobic control chips;n=4). In FIG. 7B, changes in apparent paracellular permeability (Papp)are measured by quantitating cascade blue transport across thetissue-tissue interface within the Gut Chip microdevices cultureaerobically and anaerobically (n=4).

Referring to FIGS. 8A and 8B, representative images and a graphrepresent immunofluorescence staining of nuclei and a quantification ofthe percentage of epithelial and endothelial cells. In FIG. 8A, thestaining of nuclei is with DAPI and HIF1-α in human intestinalepithelial cells and endothelial cells cultured aerobically andanaerobically (bar, 100 μm). In FIG. 8B, the graph shows thequantification of the percentage of epithelial and endothelial cellsthat expressed HIF1-α (HIF1-α⁺ cells) after exposure to the conditionsshown in a (n=3; *P<0.05, **P<0.01).

Referring to FIGS. 9A and 9B, images show a fragilis labeled with HADAand representative immunofluorescence micrographs of HADA. In FIG. 9A, acorresponding brightfield image (right) represent the fragilis labeledwith HADA before adding to chips. In FIG. 9B, the immunoflurescencemicrographs show HADA labeled Bacteorides fragilis located on top ofvillus structures when viewed from above by phase contrast imaging (bar,50 μm).

Referring to FIGS. 10A and 10B, graphs show Caco-2 viability and changesin relative abundance of quantified microbial genera. In FIG. 10A,Caco-2 viability is represented in 13 different types of media used fordefining optimized microbiota growth. In FIG. 10B, the changes arerepresentative of day 3 microbial cultures in cultured in 13 definedmedia composition. Relative abundance is determined per sample per dayas (genus read counts)/(total read counts).

Referring to FIG. 10C, a graph shows genera abundance in an originallyhuman microbiome stock derived from gnotobiotic mice (HMB) at time 0.The general abundance is significantly different than what grew out ofthe human microbiome stock derived from the gnotobiotic mice. The graphis further representative of an analysis of the diversity and relativeabundance of microbiota co-cultured in intestine chips under aerobic andaerobic conditions. Relative abundance of genera measured across allsamples highlights changes in the abundance of the different generaobserved over time, with data points representing each of threereplicate chips cultured under aerobic or anaerobic conditions at 0, 1,2 or 3 days of culture (left to right, respectively) in direct contactwith human Caco2 intestinal epithelium. Hmb indicates genera abundancein the complex microbiome stock derived from gnotobiotic mice at time 0.

Although the observed diversity and Shannon Index are lower than what isobserved in human stool samples, the graph shows an increase in richnessthat is observed compared to a starting inoculum (human biome culturedin mice) over the course of the three-day experiment. More specifically,11 well-characterized genera are identified, including Eubacterium,Oscillospira, Blautia, Sutterella, Biophila, Akkermansia, Ruminococcus,Bacteroides, Parabacteroides, Enterococcus and Citrobacter, with anadditional 8 OTUs of unknown genera from Firmicutes (5 OTUs) andProteobacteria (3 OTUs) phyla, that are present in the chips. Anobserved features indicates that some gut microbial species may growbetter under conditions that more closely mimic regions of the livingintestine than in stool. A further beneficial, important feature is thatunknown genera were present when the microbiome derived from stool wascultured on the microfluidic devices. This beneficial features indicatesthat this platform can permit the growth of species/genera that otherculture systems cannot.

Referring to FIG. 10D, a graph represents a further assessment of thephysiological mimicry obtained using the anaerobic intestine chip linedby Caco2 epithelium. Specifically, the genera identified in thisparticular study was compared with publicly available data from studiesof human stool generated by the Human Microbiome Project 34. It was notinitially expected that the composition of the microbiome grown on chipwould precisely recapitulate that of stool because the microbiome of thesmall intestine is known to show regional differences. Nevertheless, theresults show that the anaerobic culture system provides an environmentfor complex gut microbiota that sustains a diverse bacterial community,which falls into the range of abundances reported in the HumanMicrobiome Project. Furthermore, the relative abundances of the phylathat dominate the human gut, Bacteroidetes (Bacteroidetes andParabacteroides genera) and Firmicutes (Blautia, Enterococcus,Ruminococcus, and Oscillospira genera), were higher in the anaerobicchips than in the aerobic chips with some genera (Blautia andOscillospira) missing in the aerobic chips altogether.

Oxygen sensor readouts in aerobic and anaerobic chips cultured with aviable microbiome or sterilely (microbe-free) confirmed that the oxygenconcentration was maintained below 1% throughout 5-day co-culture periodin anaerobic co-cultures. Moreover, these results showed a decrease inoxygen concentration in aerobic chips cultured with microbiome overtime, which is similar to what we observed in the co-culture with B.fragilis. This was likely due to the increased vertical growth of villiobserved in these chips relative to anaerobic chips, as well as toconcomitant oxygen utilization by the bacteria, which increased innumbers by day 1 in both aerobic and anaerobic chips.

Although the oxygen concentration in the aerobic chip never reached thelow levels obtained in anaerobic chips, this decrease in oxygen likelyexplains the presence of some obligate anaerobes, such as Akkermansia,that is observed in the aerobic chips. This is surprising becausemammalian cells require oxygen while strict anaerobes find it toxic.However, it is a unique feature of the disclosed system that the systemcomponents regulate oxygen to support this co-culture. In particular,and without being bound by theory, the mammalian cells consume oxygenthat is predominantly delivered to them from their basal side to reducethe concentration on the anaerobes. In addition, and without being boundby theory, other elements of the complex microbiome, for example aerobespresent, also consume remaining oxygen that may otherwise poison theanaerobes. This is an exciting capability of the disclosed systembecause it allows the study of the interaction of anaerobes with themammalian tissue.

Interestingly, the genus Akkermansia, which has been recently implicatedas an enhancer of gut barrier function, shows a considerably highernumber of total counts in the anaerobic culture system compared to humanstool. Additionally, the genus Enterococcus is found to be present athigher levels in both chip culture systems compared to the stoolsamples, suggesting that some gut microbial species may grow betterunder conditions that more closely mimic regions of the living intestinethan in stool. Taken together, this data confirms that this anaerobichuman intestine chip system enables living human intestinal epitheliumto be co-cultured in the same channel as a complex human gut microbiomecontaining a range of bacterial genera that come much closer to what isobserved in healthy human donors than has ever been possible before.

Referring to FIG. 11 , a graph shows data representative of generagrowing or maintained in the anaerobic chip over time.

Referring to FIG. 12 , results show media tested for microbialdiversity.

Referring to FIGS. 13-15 , a bioreactor 400 shows a configuration inwhich an oxygen gradient approach is applied to a non-organ chip design.The bioreactor includes three fluidic channels 402 that are wound arounda core 404, with gas permeability properties of each layer beingconfigured to permit or block oxygen diffusion. The fluidic channels 402contain gas or liquid. According to other example, the bioreactor 400 isany other reactor configured with the oxygen gradient design describedin the present disclosure.

Referring to FIGS. 16A-16D, images and data show culture aspects forprimary human ileum chips. FIG. 16A shows the DIC microscopic image ofthe primary human ileum chips. FIG. 16B shows the confocal fluorescencemicroscopic image of the primary human ileum chips. FIG. 16C shows agraph illustrating the co-culture stably maintained five days on-chip.FIG. 16D shows a table with observed richness of various ileum samples.

Referring to FIGS. 17A-17C, villus morphology is illustrated.Specifically, FIG. 17A shows confocal fluorescence microscopic viewsillustrating the villus morphology of the primary ileal epitheliumstained for villin (cyan), F-actin (magenta), and DAPI (blue (bar, 50μm). FIG. 17B shows phase contrast views of ileum chips stained withalcian blue. FIG. 17C shows quantitation of alcian blue staining incultures shown in FIG. 17B.

Referring generally to FIGS. 18A-18C, microbiome metabolitesrecapitulate species-specific tolerance in Colon Chips. Human or mouseintestinal microbiome metabolites (e.g., isolated from specific strainsof bacterial or fecal samples incubated in a bioreactor) were added tothe intestinal channel of optically clear, human colon chips that arelined by primary human colon epithelial cells and directly opposed to asecond parallel vascular microchannel in which HIMVECs are cultured. Thetwo channels are separated by a thin, porous, ECM-coated membrane. Humanintestinal epithelium was isolated from resections or endoscopic tissuebiopsies. Endoscopic biopsies were collected from macroscopicallynormal(grossly unaffected) areas of the colon undergoing endoscopy forabdominal complaints. Organoids were grown from these tissue samples andseeded into the upper chamber of a two channel closed top microfluidicdevice. Human intestinal microvascular endothelial cells (HIMECs) wereobtained from ScienCell (Cat #2900). The intestinal luminal channelmedium was switched to 5% (vol vol−1) human (Hmm) or mouse (Mmm) gutmicrobiome metabolites isolated from PolyFermS bioreactors, diluted inphosphate-buffered saline (PBS) containing calcium and magnesium (finalosmolarity=300 mOsm kg−1), filtered through a 0.2 μm filter (Corning),and stored at −80° C.

Further referring generally to FIGS. 18A-18D, an analysis ofEHEC-induced epithelial injury on-chip is shown. Referring specificallyto FIG. 18A, representative differential interference contrast (DIC)images show the colonic epithelium in the presence of Hmm or Mmm in thepresence or absence of EHEC (bar, 100 μm). Referring specifically toFIG. 18B, pseudo-colored images show the entire colon epithelium withinthe upper channel of the colon chip (yellow or bright region) culturedin the presence of Hmm or Mmm with or without EHEC (dark regionsindicate lesion areas).

Referring specifically to FIG. 18C, quantification of epithelial lesionareas is represented based on the experimental conditions of FIGS. 18Band 18C. Epithelial lesion defined as regions in which cells normallycontained within a continuous intact epithelium have fully detached fromthe ECM-coated membrane and their neighboring cells, thus, leavingexposed regions of the membrane below. Referring specifically to FIG.18D, changes in levels of various indicated cytokines are released intothe vascular channel of the colon chips by cells cultured under theconditions described in FIGS. 18B and 18C (* p<0.05; ** p<0.01; ***p<0.001; **** p<0.0001).

Referring generally to FIGS. 19A-191 , human microbiome metabolitesstimulate bacterial motility. Referring generally to FIGS. 19A-19D,changes in the EHEC transcriptome are induced by exposure to human (Hmm)versus mouse (Mmm) gut microbiome metabolites. Referring specifically toFIG. 19A, a heat-map of differentially expressed genes (red or brighterarea indicates higher levels of expression). Referring specifically toFIG. 19B, a gene enrichment analysis is presented.

Referring specifically to FIG. 19C, a heat-map of chemotaxis andflagellar assembly pathways shows expression levels for relevantmotility-related genes in EHEC cultured in the presence of Hmm versusMmm. Referring specifically to FIG. 19D, a schematic illustrates keygenes critical in regulating chemotaxis and flagellar assembly in EHEC.Referring specifically to FIG. 19E, EHEC swimming motility tracking isillustrated (lines: bacterial movement tracks; dots: starting points forall tracked bacteria; bar, 100 μm).

Referring specifically to FIG. 19F, quantification of the fraction (%)of moving EHEC is illustrated. Referring specifically to FIG. 19G, meanvelocity of each tracked bacterium (red and black: velocity < or >3 μms−1, respectively) is illustrated. Referring specifically to FIG. 19H, adistance traveled (μm) by the moving bacteria is illustrated. Referringspecifically to FIG. 19I, Fli-C-luciferase expression levels areillustrated in medium supplemented with Hmm or Mmm (determined byquantifying area under the curve (AUC), and normalizing for the mediumcontrol) (* p<0.05; **** p<0.0001).

Referring generally to FIGS. 20A-20D, identification of specificmetabolites that mediate EHEC motility is illustrated. Referring furthergenerally to FIGS. 20A-20C, results of metabolomics analysis of humanversus mouse gut microbiome metabolites are illustrated.

Referring specifically to FIG. 20A, a Venn-diagram illustratesmetabolomics analysis workflow and total numbers of compounds identifiedin the Hmm and Mmm samples compared to the pre-fermentation medium(Pre-ferm.; label p_25: human pre-fermentation medium; label p_26 murinepre-fermentation medium). Referring specifically to FIG. 20B, a heat-mapshows 426 compounds produced by commensal bacteria that weredifferentially abundant in human (Hmm) versus mouse (Mmm) microbiomemetabolites.

Referring specifically to FIG. 20C, relative abundance shows 30microbiome metabolites that were tested (blue and red: higher levels inMmm or Hmm, respectively).

Referring specifically to FIG. 20D, results show FliC-luciferase(FliC-lux) screening for the selected metabolites (FliC-lux levels arepresented based on quantification of the AUC; grape seed oligomericproanthocyanidins (PAC) was used as a negative control; the 4 activemetabolites that induced higher FliC levels are highlighted inred/brighter color; all values were normalized against the DMSOcontrol).

Referring generally to FIGS. 21A-21C, identified active metabolitesmediate increased pathogenicity. Effect of 3,4-dimethylbenzoic acid,4-methylbenzoic acid, hexanoic acid, and heptanoic acid (4 metab.) onepithelial injury in the colon chip is in the presence or absence ofEHEC, with or without Mmm, compared to the effects of Hmm with EHEC.

Referring specifically to FIG. 21A, representative DIC images of thecolon epithelium are shown under various experimental conditions (bar,100 μm). Referring specifically to FIG. 21B, a pseudo-colored view ofthe entire epithelial layer in the colon chip (yellow or bright area) isshown under the same conditions. Referring specifically to FIG. 21C,quantification is shown of an epithelial lesion area sized underconditions shown in FIG. 21B. Epithelial lesion is defined as regions inwhich cells normally contained within a continuous intact epitheliumhave fully detached from the ECM-coated membrane and their neighboringcells, thus, leaving exposed regions of the membrane below (* p<0.05; **p<0.01).

In accordance with the disclosure provided above, the oxygen gradient isestablished across a lumen of the Gut Chip. To recapitulate aphysiologically relevant intestinal oxygen gradient profile inside OrganChips (shown in FIG. 1A), an oxygen-sensing, dual channel, human GutChip is fabricated that is composed of optically clear and flexiblepoly(dimethyl siloxane) (PDMS) polymer (FIGS. 1B and 5A), as well as ahypoxic chamber (FIGS. 6A and 6B). For real-time, non-invasive,monitoring of oxygen tension, six sensor spots containingoxygen-quenched fluorescent particles are embedded in the top and bottomportions of the Gut Chip beneath central microchannels (FIGS. 1B and5A). Changes in the fluorescent intensities of these sensors in responseto oxygen tension are captured by a Visisens camera (FIGS. 5B and 6A),and translated into oxygen concentrations by comparison with a standardOxy-4 probe system (FIG. 5C). As both the chips and sensors are composedof highly gas-permeable PDMS, the sensors respond rapidly (e.g., <30seconds) to changes in oxygen concentrations (FIG. 1C).

To simultaneously provide adequate oxygen for maintaining human cellsand an anaerobic microenvironment suitable for culturing complex humanmicrobiota while establishing a functional host-microbiome interface,the custom hypoxic chamber is flushed continually with humidified 5% CO₂in nitrogen gas (FIG. 5B). This setup enables maintaining low oxygenlevels within the lumen of the upper chamber (FIG. 1D), while theepithelium is sustained via diffusion of oxygen through the permeablePDMS membrane from the well-oxygenated medium flowing through the lowerendothelium-lined vascular channel from external oxygenated mediumreservoirs (FIG. 6B). Using this method, anaerobic conditions (<0.5%)are generated within less than 30 minutes at 243 milliliters min⁻¹ ofnitrogen flow into the hypoxic chamber (FIG. 1D). The chamber alsosustains these low oxygen levels (e.g., <5.0%) for about 15 minutesafter it is disconnected from the nitrogen source (FIG. 1D). This allowsthe chamber to be temporarily moved from the incubator for imaging orinto a bacterial glove box (e.g., to replenish culture medium or addmicrobiota) without significantly disturbing the low oxygen environment.

When human Caco-2 intestinal epithelial cells are cultured for 5 to 7days under aerobic conditions and dynamic flow, they undergo villusdifferentiation and express multiple features of the ileum portion ofthe human small intestine, including secretion of a mucus layeroverlying the apical surface of the epithelium and establishment ofbarrier function. Endothelial cells are also co-cultured on the bottomof the central porous membrane in the lower channel of the same device,where they form a hollow vascular lumen lined by cells joined by VEcadherin-containing cell-cell junctions under aerobic conditions. Theco-culture of endothelium has been shown to enhance barrier function andmucus production (e.g., expression of MUC2 and MUC5AC), as well asinfluence villi development and cytokine production by intestinal Caco2epithelium under these conditions. When Gut Chips are cultured lined bythese same two human intestinal cell types under a hypoxia gradientusing the chamber, differential interference contrast (DIC) andimmunofluorescence microscopic analysis confirms the cells again formeda villus intestinal epithelium containing polarized cells joined byZO-1-containing tight junctions (FIG. 1E, top) and a confluent HIMECmonolayer with cells linked by VE-cadherin-containing tight junctionseven under these anaerobic culture conditions (FIG. 1E, bottom). Bothcell types also remain viable under these conditions, as measured byquantifying release of the intracellular enzyme lactate dehydrogenase(LDH), which remained relatively unchanged compared to the aerobiccontrol during one week of anaerobic culture (FIG. 7A).

Measurements of apparent permeability (P_(app)) of the intestinalepithelial barrier similarly reveals no changes in the paracellularbarrier function, and these human Gut Chips display P_(app) values ofabout 1×10⁻⁷ centimeters s⁻¹ after 7 days (FIG. 7B), which are similarto those previously reported. Importantly, the present disclosureconfirms that both the human intestinal epithelium and endotheliumexperience these oxygen gradients by demonstrating that expression ofhypoxia-inducible factor 1α (HIF-1α), a key mediator of oxygenhemostasis and intestinal epithelial cell adaptation to oxygendeprivation (which is stabilized in a graded fashion in response todecreasing oxygen concentrations), is significantly higher (˜3-fold) inthe anaerobically-cultured epithelium lumen where the sensors indicate amaintenance of a hypoxic environment for up to 7 days in culture (FIG.1F), than in the adjacent oxygenated endothelium (FIGS. 8A and 8B).

The co-culture of human intestinal epithelium is disclosed below with anobligate anaerobe on-chip. Specifically, a hypoxic environment isexplored to determine if it can support co-culture of the intestinalepithelium with the obligate anaerobe, Bacteroides fragilis (B.fragilis; strain NCTC 9343), which is a human commensal symbioticbacterium that cannot grow under aerobic conditions. B. fragilisbacteria (2.5×10⁵ CFU; fluorescently labeled with HADA²⁵; FIG. 9A) isintroduced into the lumen of the intestinal epithelium-lined upperchannel (FIG. 9B) and subsequently cultured under either aerobic oranaerobic conditions, while being flushed daily to carry out CFU countsby plating. Continuous monitoring of oxygen concentration frominoculation to day 3 of co-culture reveals that the anaerobic chip setupmaintains a low oxygen environment that decreases from ˜1% oxygen levelsto 0.3% in the presence of B. fragilis (FIG. 2A). Yet, the intestinalepithelium maintains its ZO-1-containing tight junctions and apicalbrush border polarity when co-cultured in direct contact with B.fragilis under these conditions (FIG. 2B). Interestingly, the presenceof this obligate anaerobe enhances barrier function (reduced P_(app) by1.8-fold compared to aerobic conditions; FIG. 2C) after 3 days inanaerobic culture and maintains the barrier for up to 8 days in culture.As expected, the B. fragilis bacteria continues to grow in the anaerobicchips over 3 days, whereas they die off and remain at significantlylower levels under aerobic culture conditions (FIG. 2D). This dataconfirms that the hypoxic chips support the growth of an anaerobicbacterial species in direct contact with living human intestinalepithelial cells. This bacteria would have otherwise died in aconventional aerobic microfluidic system.

A mucus layers separates the commensal microbes from the epithelium. Oneof the characteristic features of host-microbiome interactions in theliving intestine is that they are mediated through an intervening mucuslayer that is secreted by the epithelium along its apical surface. Livestaining using Wheat Germ Agglutinin (WGA), which has been previouslyused for mucus visualization in vitro and in vivo, confirmed that B.fragilis resides on top of the mucus layer (FIG. 2E), which is secretedby the intestinal Caco2 epithelium. This was independently confirmed byscanning electron microscopic (SEM), which clearly revealed a continuousand dense mucus blanket that completely covered the surface of thedifferentiated villus epithelium separating it from overlying bacteriaafter 12 days of culture (FIG. 2F), much as is observed in vivo. Thiswas in contrast to SEM analysis of Caco2 intestine chips that were onlycultured for 4 days before full differentiation occurred and mucus hadaccumulated where the microvilli-lined surface of the apical epitheliumremained clearly detectable (FIG. 2F). Based on these images, thethickness of mucus layer was estimated at ˜10 μm, which is similar tothat reported with 30-day old mouse ileum.

A complex human intestinal microbiome is sustained in vitro. The hypoxicGut Chips are inoculated with a sample of complex gut microbiomeoriginally isolated from human feces, which has been stably maintainedin gnotobiotic mice (Hmb mice) in isolators for over generations. Toidentify a medium composition that would promote the growth of a complexset of commensal bacteria, the microbiome stock is first inoculated into13 different types of culture medium in standard culture tubes, then thecultures are laced in an anaerobic chamber at 37° C., and then 16s rRNAare carried out sequencing after 3 days of culture (FIG. 10A). Samplesof these 13 types of medium are also added to cultured human intestinalepithelial cells to test for toxicity (FIG. 10B). The medium thatpromotes the most diverse set of viable microbes without injuring theepithelium contains DMEM, 20% FBS, 1% glutamine, 1 mg·ml⁻¹ pectin, 1mg·ml⁻¹ mucin, 5 μg·ml⁻¹ Hemin and 0.5 μg·ml⁻¹ Vitamin K1. Themicrobiome stock is introduced into this medium (0.1 mg·ml⁻¹) andperfused through the upper epithelium-lined channel of the Gut Chipwhile oxygenated endothelial culture medium is flowed through the lowerchannel. Chips are flushed daily and 16S rRNA sequencing is carried outusing samples from the effluent of the epithelial channel to assess thebacterial diversity in each condition over 3 days of culture.

After data processing, a total of 938 OTUs are identified among allsamples, which corresponded to approximately 200 unique OTUs sharedbetween samples of each chip after filtering and removing singletons.Analysis of the alpha diversity between the two conditions shows thatthe species diversity in anaerobic chips is statistically different fromaerobic chips (PERMANOVA, p<0.001), with the trend being maintainedacross all 3 days of co-culture (FIG. 3A). Interestingly, co-culturingof these diverse microbiota under hypoxic conditions for 2 days indirect contact with the human intestinal epithelium does not compromiseintestinal barrier function, and instead, it increases barrier functionby almost 2-fold (i.e., decreases the P_(app) from 3.1×10⁻⁷ centimeterss⁻¹ to 1.6×10⁻⁷ centimeters s⁻¹ in aerobic versus anaerobic chips) (FIG.3B). In contrast, epithelial barrier function actually decreases by day3 of co-culture under aerobic conditions when co-cultured with complexgut microbiome.

To further assess the physiological mimicry obtained using the hypoxicGut Chip, the bacterial genera of the present disclosure is comparedwith publicly available data from studies of human stool generated bythe Human Microbiome Project (FIG. 3C). The results show that theanaerobic culture system provides an environment for complex gutmicrobiome that sustains a diverse bacterial community that is moresimilar to human stool than the aerobic system. The relative abundancesof the phyla Bacteroidetes and Firmicutes (Blautia, Oscillospira, andSuterella species) in the anaerobic Gut Chips are similar to thosepreviously observed in human stool, and they are all higher than thelevels detected under aerobic conditions (FIG. 3C). Interestingly,Akkermansia muciniphila, which has been recently implicated as anenhancer of gut barrier function, is more abundant in the anaerobicculture system than in stool while Parabacteroides is lower in bothculture systems indicating some gut microbial species stabilize atdifferent ratios in the Gut Chip cultures compared to stool.Nevertheless, this data confirms that this hypoxic human Gut chip systemenables living human intestinal epithelium to be co-cultured in directcontact with complex human gut microbiome containing a range ofbacterial genera that comes much closer to what is observed in healthyhuman volunteers than has ever been possible before.

To determine if the microbial communities in the anaerobic Gut Chipsystem are stable, growing, or dying during culture on-chip, theirrelative abundance is analyzed over the 3 days of co-culture with humanintestinal epithelium and underlying endothelium (FIG. 4A). It was foundthat genera composed of obligate anaerobes, such as Akkermansia,Oscillospira, Blautia, and Suterella, actually increased over time,presumably due to maintenance of low oxygen concentrations, whereasfacultative anaerobic bacterial genera, such as Enterococcus, decreased(FIG. 4A). Bacteroides, which is the highest abundance genus in theanaerobic Gut Chips, remain relatively stable over time and aremaintained at higher levels than in aerobic chips (FIGS. 4B, 4C, and 11), again confirming that the hypoxia gradient system provides apreferential environment for culture of both Bacteroides and variousFirmicutes genera.

When comparing the microbiome in the 3-day hypoxic Gut Chip co-cultureswith the microbiota cultured for a similar time in conventional liquidmedium culture in an anaerobic chamber, some genera are found to growbetter in the Gut Chip, whereas other genera displayed the oppositebehavior (FIG. 4B). Notably, Akkermansia muciniphila grows better in theanaerobic Gut Chip, presumably because the intestinal epitheliumproduces mucin, which can help fuel its growth. On the other hand, theGram-negative obligate aerobe, Citrobacter, is less abundant on-chipcompared with liquid culture. Finally, the differential abundance ofgenera is compared over time in the anaerobic versus aerobic Gut Chips.As expected, an increase is observed in abundance of obligate anaerobes,such as Sutterella, Bilophila, Blautia, Oscillospira and Akkermansia, aswell as a concomitant decrease in the abundance of Citrobacter, in theanaerobic chips compared to the aerobic chips (FIG. 4C).

The feasibility of using the present anaerobic co-culture method withpatient derived specimens was demonstrated by inoculating recentlydeveloped primary human Small Intestine-on-a-chip (Small Intestine chip)with microbiota from human fecal samples. The Small Intestine chiputilizes organoids established from intestinal biopsy specimens ortissue resections of living human intestine to create 3D intestinalvillus-like structures which exhibit epithelial barrier function,multi-lineage differentiation, enzymatic activity of brush borderenzymes and mucus production. For this study, ileal biopsies wereinitially used because this region has the highest bacteriaconcentration in the small intestine and is of interest in diseasepathologies such as Chron's and necrotizing enterocolitis. DIC andconfocal fluorescence microscopic analyses of primary human ileal chipsconfirmed the presence of a continuous, polarized, epithelial cellmonolayer with an apical F-actin-containing brush border and basalnuclei aligned along the boundary of each villin-stained extension intothe lumen of the epithelial microchannel of the chip. Fecal samples fromneonatal intensive care patients (1 mg·ml⁻¹) were introduced to theapical surface of the ileal chip in differentiation media containingmicrobial supplements while oxygenated expansion media was flowedthrough the basal channel. Chips were co-cultured with microbiota for 5days during which time they maintained epithelial barrier function (upto P_(app)˜1×10⁻⁶ cm·s⁻¹) while supporting an average bacterial richnessof 124 OTUs corresponding to 32 unique genera. While there is limiteddata on human neonatal ileum microbiota, it is likely to be less richthan the adult ileal mucosa which exhibits a richness varying from 131OTUs up to 907 OTUs. Similar studies were also carried out on duodenalchips with a lower density of bacteria (0.01 mg·ml⁻¹) to reflect thelower density of bacteria present in this segment of the small intestinein vivo. The lower optical density of the bacteria allowed for real timevisualization of bacteria surrounding villi and penetrating regionsabove crypts, which is similar to the spatial organization observed invivo.

An experimental approach is further directed to culture of freshmicrobiome with primary intestinal epithelium on-chip. This experimentalapproach is directed to co-culture complex gut microbiome obtained fromfresh human stool specimens in direct contact with primary humanintestinal epithelium (i.e., rather than using the established Caco2intestinal cell line). To do this, human intestine chips are engineeredand lined with intestinal epithelial cells isolated from organoidsderived from normal regions of surgical biopsies of human ileum, whichexhibit multi-lineage differentiation, villi formation, and mucusproduction when grown on-chip. The epithelial channels of 4 differentchips are inoculated with complex microbiome isolated from fresh humanstool samples collected from four different infants (one with acorrected gestational age of 30 weeks and three with an age of 36weeks). DIC (FIG. 16A) and confocal fluorescence microscopic (FIG. 16Band FIG. 17A) imaging of the primary human ileum chips confirmed thepresence of a villus intestinal epithelium lined by a continuouspolarized epithelium with F-actin- and villin-containing brush bordersalong its apical membrane, MUC2-producing cells, and basal nuclei. Ofnote, when production of secreted mucus is measured using alcian bluestaining (FIG. 17B), blue stained mucus is observed over the apicalsurface of the epithelium, and up to 600 ug·ml−1 of mucin is detected inthe chip outflow (FIG. 17C). As expected, the bacterial richness isreduced in the infant stool stock (586 OTUs) compared to adulthuman-derived stool (938 OTUs) at the same dilution per gram ofmaterials, and these differences in richness are accuratelyrecapitulated on-chip. The primary human intestinal epithelium isco-cultured in direct contact with this complex gut microbiome withoutcompromising epithelial barrier function, and this co-culture is stablymaintained for up to at least 5 days on-chip (FIG. 16C), much asobserved with the Caco2 epithelium. Of further mote, the microbiomecultured in these primary intestine chips also maintains a highbacterial richness, ranging from 118 to 135 OTUs (FIG. 16D)corresponding to 6 phyla (Actinobacteria, Bacteroidetes, Cyanobacteria,Firmicutes, Proteobacteria and Tenericutes) and 32 unique genera. Thus,the hypoxic intestine chip method is used to sustain a complex communityof human microbes in direct contact with normal, patient-derived, humanintestinal epithelial cells for many days in culture, which is valuablefor personalized medicine in the future.

Based on the importance of commensal gut microbiome for human health andthe lack of any in vitro model that can faithfully mimic the complexgut-microbiome interface, human Organ Chip technology is leveraged todevelop a device that enables human intestinal epithelium to beco-cultured with the highly diverse community of commensal microbes thatcomprises the human gut microbiome under aerobic and anaerobicconditions. The results show that the hypoxic human Gut Chip modeloffers a robust modular platform for recapitulating the humanintestinal-microbiome interface in vitro. Using this method, for thefirst time, it is possible to stably co-culture a complex livingmicrobiome with living mammalian cells for days in vitro. This modelaccurately recapitulates in vivo behaviors, including the maintenance ofan abundance of obligate anaerobic bacteria with ratios of Firmicutesand Bacteroidetes similar to those observed in humans feces. Thesestudies also reveal that commensal gut microbiota cultured underanaerobic conditions enhance intestinal barrier function, which is alsoconsistent with in vivo findings.

Using a custom-designed hypoxic chamber and chips containing oxygensensors that enable monitoring of local oxygen concentrations on-chip,in vivo-like oxygen gradients are recapitulated that demonstratemorphological and functional changes in the intestinal epithelium inresponse to these altered oxygen levels. When the epithelium on-chip isco-cultured with either the obligate anaerobe, Bacteroides fragilis, orcomplex human microbiome isolated from human feces under anaerobicconditions, increased bacterial growth is observed compared to aerobicconditions. This observation is further accompanied by enhancedintestinal barrier function. Importantly, providing aphysiologically-relevant oxygen microenvironment also sustains a highlevel microbial diversity (˜200 unique OTUs), increases abundance ofobligate anaerobic microbiota compared to aerobically-cultured chips,and maintains a diverse community of commensal microbe that closelyresembles that of the human gut microbiome in vivo.

Oxygen tension is one of the main regulators of intestinal function andpathogenesis of GI diseases. By integrating non-toxic oxygen sensorsinto the devices of the present disclosure, oxygen levels are measuredthroughout the microfluidic Gut Chips without interference withmicroscopy imaging, device fabrication or cell culture. Use of thesesensors, rather than incorporating multiple external oxygen-detectingprobes, enables this approach to be more easily scaled to create manyminiaturized Organ Chip platforms. The disclosed engineered hypoxicchamber also generates radial oxygen gradients across theendothelium-epithelium-microbiome interface that allows oxygenation ofthe human tissues while providing an anaerobic environment for growth ofthe obligate anaerobes. Anaerobic incubators or glove boxes are used tomaintain hypoxic conditions for bacterial cultures, but they commonlyprovide a single uniform low oxygen concentration, rather thanphysiologically-relevant oxygen gradients directed across tissue-tissueinterfaces. In contrast, the disclosed hypoxic chamber is portable,highly customizable, compatible with imaging, and most importantly,capable of engineering oxygen gradients across theendothelial-epithelial interface of any Organ Chip on demand.

Oxygen concentrations in the lumen of the human intestine are known toaffect the spatial distribution and metabolism of gut flora, and mostintestinal bacteria are obligate anaerobes that fail to grow at oxygenconcentrations greater than ˜0.5%. Any culture systems that is designedto recapitulate the host gut-microbiome interface must therefore be ableto achieve and sustain oxygen concentrations at these low levels. A pastmicrofluidic-based anaerobic culture system maintained oxygen levels aslow as 0.8% using oxygen scavengers, but this level is still too high tosupport obligate anaerobes. Using the disclosed custom hypoxic chamber,an oxygen concentration is attained that is less than 0.3% in theepithelial channel where the commensal microbes are cultured. This ismuch closer to that found in the gut lumen in vivo. Most importantly,the relevance of these hypoxic culture conditions is validated byshowing that they support the growth of the obligate anaerobe B.fragilis that cannot grow in the presence of greater than ˜0.5%dissolved oxygen, whereas most of these bacteria died off after 3 daysof in vitro culture under conventional aerobic conditions. Furthermore,the finding that co-culture of the human intestinal epithelium with B.fragilis under anaerobic conditions also increases (rather thandecreasing) intestinal barrier function on-chip is consistent with thefinding that oral delivery of B. fragilis corrects intestinalpermeability defects in a mouse autism model.

More importantly, the hypoxic human Gut Chip model supports co-cultureof complex human microbiota composed of over 200 unique OTUs and atleast 11 different genera of bacteria for at least 3 days in co-culture.Bacterial members of the Bacteroidetes and Firmicutes phyla, and to alesser degree Verrucomicrobia and Proteobacteria, which dominate humanintestinal microbiome in vivo, also dominate the disclosed Gut Chips. Inaddition, growth of other species is supported, such as Coprococcus,Anaerobacillus, Bifidobacterium, and Peptoniphilus, only in theanaerobic chips, whereas Proteobacteria that accumulate mainly at moreoxygenated regions of the proximal GI tract dominates the aerobic chips.

There remains a need to dilute the complex microbiome inoculum to avoidrapid unrestrained bacterial overgrowth. This may result in exclusion ofsome rare bacteria; however, this is ameliorated by using larger GutChips, optimizing the lumen perfusion rate, applying cyclic(peristalsis-like) mechanical deformations, or altering mediumconditions to limit bacterial overgrowth. Nevertheless, this data showsthat the anaerobic system promote more bacterial diversity than theaerobic system. Moreover, the anaerobic human Gut Chip supports a widerange of bacterial genera similar to those found in human stool, whichis much more complex than any microbiome community that has beenpreviously cultured stably for days directly in contact with mammaliancells in vitro.

Others have previously maintained complex microbiota in test tubecultures, however, the results of the present disclosure indicate thatthe presence of a more in vivo-like intestinal tissue microenvironmentsignificantly influences the composition of the microbial community. Forexample, the mucus requiring, obligate anaerobe Akkermansia muciniphilais found in higher abundance in the anaerobic gut chips containing humanintestinal epithelial cells that secrete mucus than in similarlyanaerobic liquid cultures that are artificially supplemented with mucin.In contrast to liquid cultures, the hypoxic Gut Chip also enablesidentification of effects of commensal microbes on the host epitheliumand vice versa. For example, it is interesting that the enhanced growthof Akkermansia muciniphila in the anaerobic Gut Chip is accompanied byincreased intestinal barrier function because the high abundance of thisorganism has been suggested to enhance gut barrier function in vivo.HIF-1α is also believed to control barrier integrity by regulatingmultiple barrier-protective genes, and its dysregulation may be involvedin GI disorders. Interestingly, although elevated HIF-1α expression inanaerobic Gut Chip is observed, no changes are detected in barrierfunction unless co-culturing complex microbiota.

The purpose of this disclosure is to describe an anaerobic method forco-culturing human epithelial cells with complex human microbiome in anorgan-relevant microenvironment in vitro. Although this capability isdemonstrated for the human intestine, the same methodology is applicableto study host-microbiota interactions in any Organ Chip (e.g., lung,skin, etc.). Caco2-seeded Gut Chip has been initially chosen because itnot only exhibits many functions of normal human intestine but also moreclosely resembles the ileum than other parts of the intestine. However,in aerobic condition intestinal villi grow high enough to occlude thetop channel and thus, interfere with constant medium flow and extendedco-culture periods. Because villi in the primary intestinal chips growmore slowly than the Caco2 cells, the co-cultures of complex humanmicrobiome extend for up to 5 days without compromising the epithelialviability and integrity. By integrating primary epithelial cells fromintestinal biopsies or patient-derived induced pluripotent stem (iPS)cells, as well as patient-derived microbiomes, it is expected to developpatient-, disease-, and location-specific, host-microbiome co-culturemodels. The Organ Chip technology also allows for the incorporation ofother cell types, such as immune cells and pathogens, which play crucialroles in host gut-microbiome interactions. Thus, this methodology isapplicable to unravel complex functional links between intestinalepithelial cells, immune cells, and gut microbes to understandmechanisms of human disease, discover new therapeutics, and advancepersonalized medicine.

The purpose of this disclosure is further to describe a method forco-culturing a complex living human gut microbiome, including obligateanaerobes which require strict anaerobic conditions (i.e., <0.5-1% O₂)to survive, in direct contact with human intestinal epithelial cells andtheir overlying mucus layer for extended times in vitro. Although nospecific region of the gastrointestinal system was modeled using thechips, it is noted that organ chips can be lined by cells from differentregions of the intestine (e.g., duodenum, jejunum, ileum, colon) andoxygen tensions appropriate for each region (e.g., from 5% to 0.5%moving from duodenum to colon) can be used, potentially introducing themicrobiome aspirates from each of these regions. The primary intestinechip better recapitulates the morphology, multicellular composition, andgene expression patterns of the intestinal segment from which it wasderived than other in vitro intestinal culture systems, such as theCaco2 chip and 3D intestinal organoids. Furthermore, by integratingprimary epithelial cells from intestinal biopsies as disclosed here, orpatient-derived induced pluripotent stem (iPS) cells, in combinationwith microbiomes obtained from the same patients, it is possible todevelop patient-, disease-, and location-specific, host-microbiomeco-culture models, and thus, pursue a personalized-medicine approach inthe future. That said, the Caco2 intestine chips also recapitulate manyfeatures of human intestinal physiology and pathophysiology, and thesecells can be obtained commercially (rather than requiring a patientbiopsy), which would enable their widespread use by academic andindustrial laboratories, as well as regulatory agencies (e.g., FDA).

Oxygen sensing Gut Chip manufacturing includes preparation of oxygensensor spots by mixing oxygen sensitive and optical isolating particles(PreSens GmbH, Germany) at a weight ratio of 1:1 in methanol (sigma, 50milligrams ml⁻¹) for 2 hours under constant stirring. PDMS prepolymer(Sylgard 184, Dow Corning) is added to the mixture at 1 gram ml⁻¹ andsolvent is subsequently removed by applying −70 kPa vacuum at 55° C. for2 hours. PDMS prepolymer is then mixed with a curing agent (Sylgard 184,Dow Corning) at a weight ratio of for 4 minutes under vacuum,spin-coated (150 μm thick) onto a 5 centimeter silanized silicon waferat 800 rpm for 2 minutes and cured at 60° C. for at least 30 minutes.The wafer is removed and the 150 μm thick film is punched into1-millimeter diameter sensor discs using a biopsy punch. The sensordiscs are dip-coated in an uncured PDMS (PDMS prepolymer; curing agent10:1) and embedded into the PDMS channels of the Gut Chip by placingthem in molds at the inlet, middle and outlet of both upper (epithelium)and lower (endothelium) channels, and cured in place at 60° C. for 30minutes. Gut Chip fabrication is then followed as described previously.Using this two-step molding process, these sensors are placed directlyon the surface of both the vascular and epithelial channels of the GutChips at their inlet, middle and outlet regions (FIGS. 1B and 5A). Thechip fabrication and sensor integration steps involving plasma treatmentdo not interfere with sensor function or the functionality of themicrofluidic chips (FIG. 5D), and the thickness of the sensors does notaffect the oxygen readouts when maintained between 150 to 300 μm inheight (FIGS. 5E and 5F).

Hypoxic chamber fabrication and validation includes having acrylic partscut using a laser cutter (Epilog) and assembled together with an acrylicsolvent (SciGrip Acrylic Cement). Gaskets are lasercut fromadhesive-backed silicone rubber sheets (20 Shore A hardness,McMaster-Carr) and magnetic clasps are attached using adhesive backedmagnets. The hypoxic chamber is tested using a calibrated Oxy-4 opticalprobe system (PreSens GmbH, Germany) to verify the hypoxic conditions.To do so, the chamber is purged with 5% CO₂ in N₂ bubbled throughdeionized water at 81 mL min⁻¹, 162 mL min⁻¹, or 243 mL min⁻¹ for 1 h atwhich point N₂ flow is stopped and the chamber allowed (3 h) to recoverto atmospheric oxygen.

Oxygen sensing in the Gut Chip includes visualizing and quantifying theconcentration of oxygen throughout the chip. Oxygen measurements areperformed through non-invasive fluorescence read-out usingVisiSens-system (PreSens GmbH, Germany). Using a CCD-camera and theVisiSens software (V1.1.2.10), oxygen amount is detected at sensor spotsand displayed using a computer code in pseudo colors. The software isdesigned to calculate oxygen levels on the sensor spots via calibrationof fluorescence reading with defined oxygen levels at 0 and 100% airsaturation (i.e., 20.9% O₂ of all dissolved gas by volume). In allexperiments, oxygen levels are quantified after comparing the readingswith the calibration values. Air-saturated water and oxygen-freesolution (Oakton, WD-00653-00) are used to calibrate the sensor spots.Because the field-of-view of the VisiSens camera is inherently small, alinear positioning system is designed (FIG. 5A) that positions thecamera directly beneath the Gut Chips in the hypoxia chamber (FIG. 6A).This allows indexed motions of the camera to any sensor spot along thechip or between the chips and thus, facilitates reproducibly imagingmultiple chips in one run. The sensors do not obscure regular imaging ofthe chips as they only cover a small portion of the culture area (˜3mm²), allowing for regular monitoring of cultures throughout theexperiment. A black opaque box is designed to cover the entire chipculture chamber and VisiSens camera, for blocking extraneous light. Toanalyze the accuracy of sensor spots inside the chips, custom gasmixtures are used with known oxygen concentration, i.e., 0, 1, and 12.5%O₂. The VisiSens imaging system is validated using an Oxy-4 opticalprobe system (PreSens) with optical fibers (POF-L2.5, PreSens, Germany).

For oxygen sensor analysis, images of oxygen sensors are processed inMATLAB (Mathworks). The images are binarized using Otsu's method.Morphological erosion and dilation is preformed to eliminate anyspurious artifacts created during binarization. Simulated annealing isapplied to find the correct assignment of sensors in each imageregardless of the chip alignment. The sum of the distance of each of thesensor's centroids in the current image between the nearest sensor'scentroid in the original image is minimized. After aligning the images,the sensors in the current image are registered consistently with thesensors in the former image, and colorimetric analyses are computed. Theaverage intensities are calculated for each of the red, blue, and greenchannels, in each sensor. The uncalibrated signal from each sensor istaken to be the average green intensity divided by the average redintensity. The uncalibrated signal is then fit to a calibration curve.

A modified Michaelis-Menten two-point calibration is used as the mostgeneralizable model,C_(oxy)=k_(min)+(k_(max)−k_(min))×[x_(g:r)/(k_(rate)+X_(g:r))];k_(max)=a×k_(atm), where x_(g:r) denotes the ratio of average greenintensity to average red intensity, C_(oxy) is the fraction ofatmospheric oxygen, k_(min) is the sensor signal at anaerobicconditions, k_(max) is the sensor signal when saturated with oxygen, andthe concentration of oxygen is given as C_(oxy). k_(rate) explains theeffect that the observed signal, x_(g:r), has on the concentration ofoxygen. The atmospheric oxygen concentration does not fully saturate thesensor with oxygen. To overcome this, actual maximum possible signalfrom a sensor, k_(max), is estimated by multiplying the uncalibratedsignal at atmospheric concentration, k_(atm), by a scale factor α. TheMichaelis-Menton curve is approximately linear between x_(g:r)=k_(atm)and x_(g:r)=k_(max), scaling by a linear coefficient does not hamper theequation's ability to generalize between sensors. The curve is fit usingimages acquired at known oxygen concentrations. The known concentrationsare measured by Oxy-4 optical probe system (PreSens GmbH, Germany). Theoxygen concentrations is also validated by flowing oxygen at knownconcentrations over the probe and sensor. Both k_(rate) and α are fitusing the data. The model produces a suitable fit for the data (R²=0.990training, R²=0.997 and 0.998 for testing) (FIG. 6A). The fitted modelgeneralized well for trials is repeated in different chips and ondifferent days (FIG. 6B).

For cell culture procedures, prior to cell seeding, microfluidic sensorchips are activated using oxygen plasma (Diener ATTO) and functionalizedwith (3-Aminopropyl) trimethoxysilane (Sigma, 281778) as reportedpreviously. Chips are then washed with ethanol, oven-dried at 80° C. andcoated with 30 μg ml⁻¹ Collagen (Gibco, A10483-01) and 100 μg ml⁻¹Matrigel (BD Biosciences, 356237) in the serum-free Dulbecco's ModifiedEagle Medium (DMEM; Gibco, 10564011) for 1 hour at 37° C. Afterwards,human intestinal microvascular endothelial cells (HIMECs; ScienCell) areseeded (1.5×10⁵ cells cm⁻²) in the bottom channel of the chips, onopposite side of the porous membrane. Chips are then placed in a 37° C.incubator for 1.5 hours. For HIMECs culture, endothelial growth medium(EGM2-MV) containing human epidermal growth factor, hydrocortisone,vascular endothelial growth factor, human fibroblastic growth factor-B,R3-Insulin-like Growth Factor-1, Ascorbic Acid and 5% fetal bovine serum(Lonza Cat. no. CC-3202) is used.

Human intestinal epithelial cells (Caco2 BBE human colorectal carcinomacell, Harvard Digestive Disease Center) are then seeded into the topmicrochannel of the chip (1.5×10⁵ cells cm⁻²) and incubated for 1.5hours. Epithelial cells are fed with DMEM (Gibco, 10564011) containingPen/Strep and 20% Fetal Bovine Serum (FBS; Gibco, 10082-147). Afterwashing with 200 μl of medium, chips are cultured statically overnightto allow cells to form monolayers on both sides of the membrane. A dayafter seeding, top and bottom channels are perfused (60 μL h⁻¹) withepithelial medium and reduced-FBS endothelial medium, respectively.Chips are kept in this condition until villus-like intestinal epitheliumspontaneously appears. For anaerobic culture, the same procedure isfollowed except that after 1 day of perfusion in aerobic conditions,chips are placed in a hypoxic chamber and continuously perfused with 5%CO₂ in N₂ flowed at 243 mL min⁻¹.

Referring to organoid culture procedure, for human intestinal organoids,de-identified endoscopic tissue biopsies were collected from grosslyunaffected (macroscopically normal) areas of the ileum and duodenum in10-14-year-old patients undergoing endoscopy for gastrointestinalcomplaints. Informed consent and developmentally-appropriate assent wereobtained at Boston Children's Hospital from the donors' guardian and thedonor, respectively. All methods were carried out in accordance with theInstitutional Review Board of Boston Children's Hospital (Protocolnumber IRB-P00000529) approval. Tissue was digested in 2 mg·ml⁻¹collagenase I for 40 min at 37° C. followed by mechanical dissociation,and isolated crypts were re-suspended in growth factor-reduced Matrigel(Becton Dickinson) and polymerized at 37° C. Organoids were grown inexpansion medium (EM) consisting of Advanced DMEM/F12 supplemented withL-WRN conditioned medium (50% v/v, ATCC), glutamax, HEPES, murineepidermal growth factor (50 ng·ml⁻¹), N₂ supplement, B27 supplement,human [Leu15]-gastrin I (10 nM), n-acetyl cysteine (1 mM), nicotinamide(10 mM), SB202190 (10 μM) and A83-01 (500 nM). Differentiation medium(DM) is EM without L-WRN conditioned medium, nicotinamide and SB202190,but supplemented with human recombinant R-spondin 1 (Peprotech; 1μg·ml⁻¹), human recombinant Noggin (Peprotech; 100 ng·ml⁻¹) andγ-secretase inhibitor DAPT (10 μM). Organoids were passaged periodicallyby incubating in Cell Recovery Solution for 40 min at 4° C., followed bymechanical dissociation. Organoids were seeded on chips between passagenumber 5 and 25.

Referring to primary small intestine chip culture, microfluidic chipswere obtained from Emulate Inc. (Boston, MA). Chips were chemicallyactivated using Emulate ER1 and ER2 solutions. Type I collagen (200μg·ml⁻¹) and Matrigel (1% in PBS) were then introduced into thechannels, and incubated in a humidified 37° C. incubator for 2 h beforewashing with PBS. Epithelial organoids were isolated from Matrigel andthe cells dissociated with TrypLE supplemented with 10 μM Y-27632.Epithelial cells were then re-suspended in EM (6×10⁶ cells·ml⁻¹; ofwhich 30 μl is used to fill the apical chamber of each chip resulting in˜180,000 cells·chip⁻¹), infused into the top channel, and incubatedovernight in static at 37° C. The following day EM was perfused at 60μl·h⁻¹ through the top and bottom channels and a peristalsis-likestretch (10% cell strain, 0.15 Hz frequency) was applied using a vacuumpump controlled by an electronic vacuum regulator (ITV009, SMC Corp.)and an Arduino microcontroller. Chips were maintained under theseconditions until the visual development of villus like structures (˜14days). The apical media was then replaced with antibiotic free DMcontaining microbial supplements (1 mg·ml⁻¹ pectin, 1 mg·ml⁻¹ mucin, 5μg·ml⁻¹ Hemin and 0.5 μg·ml⁻¹ Vitamin K1) and the basal media wasreplaced with antibiotic free EM.

Bacterial and microbiota culture includes B. fragilis (9343 strain)grown overnight at 37° C. under anaerobic conditions (80% N₂, 10% Hz,10% CO₂) in rich media containing yeast extract (5 g L⁻¹), proteasepeptone (20 g L⁻¹), NaCl (5 g L⁻¹), hemin (5 mg L⁻¹), vitamin K1 (0.5 mgL⁻¹), K₂HPO₄ (5 g L⁻¹) and HADA (HCC-amino-D-alanine, λ_(em)˜450 nm; 0.8mM). Hemin, vitamin K1, K₂HPO₄, and HADA 25 are added through a 0.22 μmfilter after autoclaving the other ingredients. B. fragilis is pelletedat 5000 g, washed once in DMEM, and re-suspended in Caco2 media (DMEM20% FBS, 1% glutamine, 1 mg ml⁻¹ pectin, 1 mg ml⁻¹ mucin, 5 μg ml⁻¹Hemin, 0.5 μg ml⁻¹ Vitamin K1) at 1×10⁷ CFU ml⁻¹. For microbiotaco-culture, colon and cecum content from five mice colonized withhealthy human microbiota¹⁸ is collected and re-suspended in sterile PBSinside an anaerobic chamber (100 mg of content ml⁻¹). The slurry is thenfiltered (40 μm) and aliquoted and stored at −80° C. as the humanmicrobiome stock, which is diluted 1:100 in epithelial medium when addedto Gut Chips. For microbiota co-culture with patient-derived specimens,fecal samples were collected from infants born at Brigham and Women'sHospital in Boston, MA and cared for in a single-center NewbornIntensive Care Unit (NICU). Parental consent was obtained and all studyprocedures followed a protocol that was approved by the Partner's HumanResearch Committee for Brigham and Women's Hospital and MassachusettsGeneral Hospital (Protocol number 2012-P-002453). Fecal samples werecollected from preterm infants born prior to 32 weeks of gestation frombirth until discharge. Briefly, diapers with fecal samples werecollected daily by the bedside nurse, placed in a specimen bag, andstored at 4° C. for no more than 24 hours. Fecal material was extractedfrom diapers using sterile procedures and immediately frozen at −80° C.Selected samples were suspended in Brain Heart Infusion media (100mg·ml⁻¹) to create a stock solution.

Gut-microbiota co-culture in Gut Chips includes washing media reservoirswith PBS 24 hours before adding bacteria. Antibiotic-free media is thenadded to Gut Chips in a tissue culture hood (aerobic conditions) or inan anaerobic chamber (anaerobic conditions). The next day, 25 μl of B.fragilis (1×10⁷ CFU ml⁻¹) or microbiota stock (1:100) is added to theapical side of differentiated Gut Chips in a tissue culture hood(aerobic conditions) or in an anaerobic chamber (anaerobic conditions).Chips are left static for 30 minutes and then perfused at 1 μl min⁻¹.Every 24 hours, a 2-minute flush at 50 μl min⁻¹ is performed and theflush outflow is collected and serial dilutions are plated on Brucellaplates incubated at 37° C. in an anaerobic chamber (B. fragiliscultures) or sent to Diversigen, Inc. (complex microbiota cultures) for16S rRNA sequencing.

Morphological analyses include, for each experiment, analysis of 3independent gut chip samples at each interval. The intestinal epitheliumvillus structures are evaluated using differential interface contrast(DIC) microscopy (Zeiss Axio Observer Z1 2, AXIO2). Immunoflorescencemicroscopy with a laser scanning confocal microscopes (Leica SP5 X MPDMI-6000 and Zeiss TIRF/LSM 710) is used to study the villusmicroarchitecture. High-resolution horizontal or verticalcross-sectional images are obtained using deconvolution (Huygens)followed by a 2D projection process. IMARIS (MARIS 7.6 F1 workstation;Bitplane Scientific Software) and ImageJ are used for analyzing theobtained images.

For immunofluorescence microscopy, epithelial and endothelial cells arewashed with PBS, fixed with paraformaldehyde (20 min; PFA, 4%; ElectronMicroscopy Sciences, 157-4) and subsequently washed with additional PBS.Permeabilization of cells is done with Triton X-100 (20 minutes; 0.25%;Sigma, T8787), followed by incubation in blocking buffer containing 1%BSA (Sigma, A4503) and 10% donkey serum (Sigma, D9663) for 30 minutes atroom temperature. Primary antibodies against ZO1 (Life Technologies,33-9100, dilution 1:200), VE-cadherin/CD144 (BD Biosciences, 555661,dilution 1:200), Villin (Life Technologies, PAS-29078, dilution 1:100),HIF-1α (Abcam, ab16066, dilution 1:100) or Cleaved Caspase-3 (Cas-3,Cell Signaling, 9661, dilution 1:100) are added and incubated overnightat 4° C., followed by 6 PBS washes (5 min each).

Cells are then incubated with secondary antibodies (Life Technologies)for 1 hour at room temperature and washed with PBS afterwards. Cells areco-stained with DAPI (Invitrogen, D1306). For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL)immunostaining, Click-iT TUNEL Alexa Fluor Assay Kit (Invitrogen,C10247) is used according to the manufacturer's protocol. Chips areco-stained with DAPI (Invitrogen, D1306) as the nuclear DNA marker.Apoptotic cells are counted from 20 different fields (10 fields eachfrom 2 replicates) to get an average number of TUNEL- and Cas-3-positivecells per field. To induce apoptosis, chips are treated with 1 unit ofDNase I solution for 30 min at room temperature. Microscopy is performedwith a laser scanning confocal microscope (Leica SP5 X MP DMI-6000 orZeiss TIRF/LSM 710).

Referring to mucus detection and visualization, Wheat Germ Agglutinin(WGA) Alexa Fluor 488 conjugate (Thermo Fisher Scientific) was used forlive cell imaging. Briefly, WGA solution (25 μg·ml⁻¹ in culture medium)was flowed through the epithelium channel for min. The top channel waswashed subsequently with PBS in the dark and counter-stained with DAPIto visualize nuclei. To stain acidic mucopolysaccharides within theintestinal mucus, intestine chips were stained with 0.1% (w/v) alcianblue solution (pH 2.5; 8GX, Sigma) in 3% acetic acid (Sigma) by flowingthe solution into the microchannels at 50 μL·h⁻¹ for 12 h, and thenwashing with PBS.

Referring to paracellular permeability measurements, 50 μg ml⁻¹ ofcascade blue (5.9 kDa; ThermoFisher, C687) are introduced to theepithelium channel (60 mL hr⁻¹) and fluorescence intensity (390 nm/420nm) of top and bottom channel effluents are measured using a multi-modeplate reader (BioTek NEO). Apical-to-basolateral flux of theparacellular marker is calculated based on the following equation:P_(app)=(dQ/dt)/A·dC. P_(app) (cm s⁻¹) denotes the apparent permeabilitycoefficient, dQ/dt (g s⁻¹) is molecular flux, A (cm²) is the total areaof diffusion and dC (mg mL⁻¹) is the average gradient.

Referring to cellular toxicity, CytoTox 96 Non-Radioactive CytotoxicityAssay (LDH; Promega, G1780) is used according to the manufacturer'sinstructions to measure epithelium and endothelium death rate atdifferent intervals in both aerobic and anaerobic culture conditions.Effluents are collected from top and bottom channels, mixed with LDHsubstrate reagent and incubated for 30 minutes. The enzymatic reactionis terminated using stop solution (containing acetic acid) and theabsorbance at 492 nm is recorded using a multi-mode plate reader (BioTekNEO). The LDH activity is assessed using quadruplicate of each group,calculated after subtracting the background absorbance values andreported as a fold change of the total LDH values of control group.

Referring to rRNA sequencing analysis, raw reads are analyzed usingQIIME 1.0 under standard protocols and resulting joined reads arealigned to the Greengenes database. A total of 938 operational taxonomyunits (OTUs) are identified. As one of the steps in the disclosedanalyses of the 16S sequencing data, OTUs that did not meet certaincriteria in terms of representation across all the samples are removed.The data is loaded into R and the phyloseq package is used for furtherprocessing. After performing diversity analyses, all singletons areremoved from the data set and the OTUs are summarized to the genuslevel, resulting in a total 42 unique genera. Differential abundance ofthese genera between the two culture conditions, i.e., aerobic andanaerobic, is done using the DESeq2 package. OTUs showing a differentialabundance with an FDR corrected p-value q<0.05 are consideredsignificant. The PERMANOVA test is run in R using the adonis function inthe vegan package between aerobic and anaerobic conditions, as well asbetween the two oxygen conditions across the different days.

Referring to statistical analysis, all experiments are carried out atn=3-6 (see captions of respective figures), and results and error barsindicate mean±standard error of the mean (s.e.m). Data analysis isperformed with a one-way analysis of variance (ANOVA) with Tukey HSDpost hoc tests using Graphpad Prism software. Statistical analysisbetween two conditions is performed by an unpaired student's t-test. Pvalues of less than 0.05 are considered to be statistically significant(*P<0.05, **P<0.01, ***P<0.001).

According to other embodiments, a two-channel design is expanded tobioreactors, including a parallel plate reactor or a rolled-up platereactor. One of the benefits of the oxygen gradient, which enablesco-culture of mammalian and bacterial cells, is the permeability of themembrane and the top of the device to oxygen. As such, the oxygennecessary for human cells is delivered from the bottom channel but,then, it is consumed by cells or it quickly diffuses-out through the topchannel Chip body and through the perfuse channel itself. This aspect ismaintained in large systems as long as there are two or more chambers orchannels that enable the oxygen flux path.

According to other aspects of the present disclosure, it is furtherdisclosed that endoscopic analysis of human patients infected with EHEChas revealed acute inflammation of the colon and ex vivo infectionexperiments similarly demonstrated colonization as well as attaching andeffacing (A/E) lesions in human colonic biopsies. Humans are susceptibleto EHEC infection at a very low dose (102) whereas the dose required toinduce infections in mice is 100,000-fold higher.

Surprisingly, the present disclosed studies have discovered that humanmicrobiome metabolites increased enterohemorrhagic Escherichia coli(EHEC)'s ability to induce epithelial damage. In fact, greaterepithelial injury is observed when human metabolites are present, whileEHEC does not induce lesion formation in the absence of microbiomemetabolites. Epithelial damage is also associated with an increase inexpression of EHEC genes related to known virulence pathways related tochemotaxis and motility.

In contrast, mouse microbiome metabolite product protects against thedamaging effects of this infectious pathogen. Thus, in some embodiments,metabolites from samples of human gut biomes enhance epithelial injuryduring pathogenic bacteria infections of the gut.

Human microbiome metabolites including 4-methyl benzoic acid,3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid, addedindividually to a colon-chip enhance epithelial injury during a EHECinfection of the chip. Moreover, addition of these four identified humanmicrobiome metabolites is sufficient to convert the tolerant murinemicrobiome phenotype into an injury response that mimics than producedby addition of the human microbiome products.

For example, on day 8 of a colon chip culture, the luminal culturemedium is replaced with the same medium supplemented with human ormurine microbiome metabolites (diluted 1:20 in a PBS-water basedsolution to 300 mOsm kg−1), while continuing to flow the sameendothelial culture medium through the vascular channel. Perfusion iscontinued for 24 hours, followed by introduction of EHEC (1.7×105;serotype O157:H7) into the apical lumen in the same medium for 3 hoursunder static conditions to allow for bacterial cell attachment; mediumflow is then re-established and continued for 24 additional hours.

Although metabolic analysis is used to pursue the mechanism by which Hmmand Mmm produce different effects on EHEC-induced epithelial injury, thefocus is on known metabolites because these compounds could be obtainedcommercially and tested experimentally to validate their effects. Otherunknown microbiome-derived metabolites present in the Hmm sample mayhave additional modulating activities, which could be explored in thefuture using fractionation of the Hmm sample and in-depth massspectrometry analysis.

A similar experimental approach can identify microbiome-derivedmodulators of other enteropathogens that exhibit species-specificdifferences in pathogenicity in the future. Further, the methodsdescribed herein are contemplated to offer new mechanistic insights intowhy certain individuals or species are more tolerant to specificinfectious pathogens than others.

TABLE 1 List of exemplary 30 known metabolites enriched in Hmm comparedto Mmm that are selected for fliC -luciferase screening (CAS n: ChemicalAbstracts Service number; “name”: metabolites with a known name;“similarity”: closest MSMS spectrum in the reference database to the tothe analyte, with a 95% confidence in identification). Compound Name ID(weight_retention time) CAS n Identification DiHome 260.19857_2.219263399-35-5 similarity 2-Hydroxyhexanoic acid 132.07856_2.024 6064-63-7name 2-Methylbenzoic acid 272.10498_3.508 118-90-1 similarity2-sec-Butyl-3-methoxypyrazin 371.17888_4.51 24168-70-5 similarity3,4-Dimethylbenzoic acid 150.06795_2.412 619-04-5 name4-Dodecylbenzenesulfonic acid 326.22039_2.728 121-65-3 similarity4-Methylbenzoic acid 274.18934_2.471 99-94-5 similarity Acetylarginine114.07927_3.982 155-84-0 similarity Glutamine 304.17409_7.141 5959-95-5similarity Glucosamine 143.0943_9.55 66-84-2 similarity Deoxycholic acid392.2928_2.451 83-44-3 name DL-Arginine 174.11135_8.207 7200-25-1similarity DL-Homoserine 100.01609_3.117 1927-25-9 similarityDocosahexaenoic acid ethyl ester 409.31776_2.465 84494-72-4 similarityHeptanoic acid 130.09938_2.145 111-14-8 name Hexanoic acid116.08368_2.286 142-62-1 similarity Isoleucine 187.13167_9.36 73-32-5similarity Kanosamine 160.08446_6.8 57649-10-2 similarity L-Lysine146.12996_13.781 56-87-1 similarity L-Tyrosine methyl ester195.13668_4.829 1080-06-4 similarity Methyl-L-histidinate 169.0847_5.059332-80-9 similarity N-Acetyl-L-methionine 175.08435_4.076 65-82-7similarity N-Acetyl-L-phenylalanine 207.0894_5.157 2018-61-3 nameN-Acetylhistamine 153.0898_7.032 673-49-4 name Pimelic acid116.12072_2.346 111-16-0 similarity Prolylleucine 228.15808_6.39661596-47-2 similarity Pyridoxine 168.98662_5.97 58-56-0 similaritySilibinin 178.11017_3.861 22888-70-6 similarity UDP-N-acetylglucosamine98.04821_6.285 91183-98-1 similarity Uracil 111.99201_6.581 66-22-8 nameDimethyl sulfoxide (DMSO) NA 67-68-5 NA Proanthocyanidin (PAC) NA222838-60-0 NA

The following are exemplary materials and methods. Bioreactor culturesinclude soluble metabolites isolated from bioreactor cultures of complexpopulations of murine or human intestinalcommensal microbes. umanmicrobiome metabolites (Hmm) or mouse microbiome metabolites (Mmm) arecollected from PolyFermS continuous intestinal fermentation bioreactorsin which complex mouse or human microbiome samples are cultured for twoweeks under conditions that mimic the internal milieu of the largeintestine; the commensal bacterial content of the cultures was definedat the phylum and genus levels using 16S rRNA gene sequencing.

For metabolomics, samples are centrifuged at 10,000×g for 5 min followedby biphasic chloroform-methanol extraction. All samples are run foruntargeted mass spectrometry on a ThermoFisher Q-exactive massspectrometer. Compound Discovery Software is utilized to assign compoundnames (95% confidence). If the parent ion is not found, the compoundwith the closest spectrum is used as an identifier, thus indicating apotential substructure of the original metabolite. In the case ofmultiple metabolites matching to the same identifier, priority is givento the metabolite identified with the highest average area value. In oneanalysis, 426 metabolites are identified enriched in either Hmm or Mmm,and all the metabolites with an assigned compound name are selected.Within these metabolites, all 30 commercially available compounds areselected, while known synthetic prescription drugs, antimicrobialagents, or potential chemical contaminants (Table 1, examples) areexcluded and screened them for their effect on EHEC flagellar motility.Some readouts include the following: 16S rRNA gene sequencing, usingknown methods; bacterial motility tracking; fliC-luciferase reporterassay; genomic DNA analysis, e.g. in biomes before, during and afterincubation in a PolyFermS device, before, during (collected fromeffluent) and after incubation on-chip.

Referring to colon chip infection, colon chips were cultured in theintestinal lumen channel of a chip in 5% (vol vol−1) human or mouse gutmicrobiome metabolites isolated from PolyFermS bioreactors, diluted inphosphate-buffered saline (PBS; final osmolarity=300 mOsm kg−1) or 24hours. The following day, the intestinal channel was infected with1.7×105. EHEC-GFP or EHEC Δ fliC (both generated from NR-3 E.coli/EDL931; serotype O157:H7), by adding the bacteria into the channellumen in medium again with or without Hmm or Mmm. Chips were maintainedunder static conditions for 3 hours to promote EHEC colonization, andthen perfused at 60 μl h−1.

For epithelial lesion analysis, one day post-infection, colon chips werewashed with PBS and fixed with 4% paraformaldehyde in PBS for 2 hours.The chips were imaged using a Leica DM IL LED microscope and images werestitched together with Basler Phylon Software. The area occupied bycells and the total area of the chip were measured using Fiji software.

For bacteria viability, bacteria were grown 6 hours at 37° C. in medium,in some embodiments, containing Hmm or Mmm, then propidium iodidesolution was added at a final concentration of 10 mg ml−1 for 5 min atroom temperature.

For bacteria swimming plate assay, swimming motility was assessed using0.25% agar LB plates. Overnight cultures of EHEC or EHEC-GFP bacteriawere standardized at 1 OD600 and 1.5 μl of the culture medium was addedto the center of the agar plate with a sterile pipette tip. Bacterialswimming was quantified at 12 hours, imaging the plates using aFluorChem M imaging system (ProteinSimple). The area occupied bybacteria was then measured using Fiji.

TABLE 2 Exemplary Reagents and resources. REAGENT OR RESOURCE SOURCEIDENTIFIER Bacterial Strains NR-3 Escherichia coli, EDL931, serotype beiResources EDL931 (Serotype O157:H7 O157:H7) NR-96 Escherichia coli, B2F1(Serotype bei Resources B2F1 (Serotype O91:H21) O91:H21) EHECfliC-luciferase (Serotype O157:H7) This study EHEC fliC-luciferase(serotype O91:H21) This study EHEC-GFP This study EHEC ΔfliC This studyExperimental Models: Cell Lines Human Intestinal Microvascular ScienCellCat#2900 Endothelial Cells (HIMEC) Human Colonic Organoids L-WRN ATCCCat#CRL-3276 Chemicals, Peptides, recombinant proteins Pectin (citrus)Sigma-Aldrich Cat#P9135 Xylan (beechwood) Chemie BrunschwigCat#APOBI3856 AG Arabinogalactan (larch) Lonza Cat#189452 Guar gumSigma-Aldrich Cat#G4129 Inulin Cosucra Cat#FIBRULOSE-F97 Soluble potatostarch Sigma-Aldrich Cat#S2004-1KG Soluble corn starch Sigma-AldrichCat#S9679 Mucine Sigma-Aldrich Cat#M2378 Casein acid hydrolysateSigma-Aldrich Cat#A2427 Peptone water Thermo Fisher Cat#CM0009BDiagnostics AG Bacto tryptone Becton Dickinson Cat#211705 Yeast extractVWR International Cat#1.11926.1000 L-cysteine HCl Sigma-AldrichCat#W778567 Bile salts Thermo Fisher Cat#LP0055J Diagnostics AG KH2PO4VWR International Cat#26923.298 NaHCO3 Sigma-Aldrich Cat#13433 NaCl VWRinternational Cat#1000152 KCl Sigma-Aldrich Cat#12636 MgSO4 anhydratedSigma-Aldrich Cat#63140 CaCl2*2 H2O Sigma-Aldrich Cat#1000039 MnCl2* 4H2O Sigma-Aldrich Cat#63536 FeSO4* 7H20 Sigma-Aldrich Cat#12354 HeminSigma-Aldrich Cat#H9039 Tween 80 Sigma-Aldrich Cat#P8074 Pyridoxine-HCl(Vit. B6) VWR International Cat# A8093.0025 4-Aminobenzoic acid (PABA)Sigma-Aldrich Cat#A9878 Nicotinic acid (Vit. B3) Sigma-AldrichCat#N4126-100G Biotine Sigma-Aldrich Cat#14400 Folic acid VWRInternational Cat#A2085.0010 Cyanocobalamin Sigma-Aldrich Cat#V2876Thiamine Sigma-Aldrich Cat#T4625 Riboflavin Sigma-Aldrich Cat#R4500Phylloquinone Sigma-Aldrich Cat#95271-1G Menadione VWR InternationalCat#ICNA0210225925 Pantothenate Sigma-Aldrich Cat#P2250 AdvancedDMEM/F12 Thermo Fisher Cat#12634-010 Scientific GlutaMAX Thermo FisherCat#35050-061 Scientific HEPES Thermo Fisher Cat#15630-106 ScientificB27 supplement Thermo Fisher Cat#17504-044 Scientific N2 supplementThermo Fisher Cat#17502-048 Scientific Nicotinamide Sigma-AldrichCat#N0636 N-acetyl-1-cysteine Sigma-Aldrich Cat#A5099 [Leu15]-gastrin I,human Sigma-Aldrich Cat#G9145 Recombinant murine epidermal growthPeprotech Cat#315-09 factor Recombinant murine Noggin PeprotechCat#250-38 Recombinant murine R-Spondin-1 Peprotech Cat#315-32Recombinant Murine Wnt-3a Peprotech Cat#315-20 Activin-like kinase (ALK)inhibitor (A83- Tocris Cat#2939 01) p38 Mitogen-activated kinase (MAPK)Sigma-Aldrich Cat#S7067 inhibitor (SB202190) Rho-associated proteinkinase (ROCK) Sigma-Aldrich Cat#Y0503 inhibitor MicrovascularEndothelial Cell Growth Lonza Cat#CC3202 Medium-2 BulletKit (EGM-2MV)Human epidermal growth factor Lonza Cat#CC3202 Vascular endothelialgrowth factor Lonza Cat#CC3202 Human fibroblastic growth factor-B LonzaCat#CC3202 R3-Insulin-like Growth Factor-1 Lonza Cat#CC3202 AscorbicAcid Lonza Cat#CC3202 Primocin InvivoGen Cat#ant-pm-1 Bacto Tryptone BDBiosciences Cat#211699 Bacto Yeast Extract BD Biosciences Cat#212720Sodium chloride HAWKINS PC Cat#10142-840 RPMI Medium 1640 LifeTechnologies Cat#72400-120 Grape seeds oligomeric proanthocyanidinsSigma-Aldrich Cat#1298208 (PAC) Dimethyl sulfoxide (DMSO) Sigma-AldrichCat#D2650 DiHome Cayman Chemicals Cat#10009832 2-Hydroxyhexanoic acidMedChem Express Cat#HY-75954 2-Methylbenzoic acid MedChem ExpressCat#HY-41494 2-sec-Butyl-3-methoxypyrazin MedChem Express Cat#HY-W0171403,4-Dimethylbenzoic acid MedChem Express Cat#HY-W017434 and andSigma-Aldrich D149403 4-Dodecylbenzenesulfonic acid MedChem ExpressCat#HY-23059 4-Methylbenzoic acid MedChem Express Cat#HY-76547 andSigma-Aldrich and T36803 Acetylarginine MedChem Express Cat#HY-W014130Glutamine MedChem Express Cat#HY-100587 Glucosamine MedChem ExpressCat#HY-N0733 Deoxycholic acid MedChem Express Cat#HY-N0593 DL-ArginineMedChem Express Cat#HY-N0454 DL-Homoserine MedChem ExpressCat#HY-W012870 Docosahexaenoic acid ethyl ester MedChem ExpressCat#HY-W011120 Heptanoic acid MedChem Express Cat#HY-42935 and andSigma-Aldrich 75190 Hexanoic acid MedChem Express Cat#HY-N4078 andSigma-Aldrich and 153745 Isoleucine MedChem Express Cat#HY-N0771Kanosamine MedChem Express Cat#HY-112176 L-Lysine MedChem ExpressCat#HY-N0469 L-Tyrosine methyl ester MedChem Express Cat#HY-W007671Methyl-L-histidinate MedChem Express Cat#HY-W017006N-Acetyl-L-methionine MedChem Express Cat#HY-W012499N-Acetyl-L-phenylalanine MedChem Express Cat#HY-Y0068 N-AcetylhistamineMedChem Express Cat#HY-112175 Pimelic acid MedChem Express Cat#HY-Y1139Prolylleucine MedChem Express Cat#HY-112173 Pyridoxine MedChem ExpressCat#HY-N0682 Silibinin MedChem Express Cat#HY-13748UDP-N-acetylglucosamine MedChem Express Cat#HY-112174 Uracil MedChemExpress Cat#HY-I0960 4% Paraformaldehyde Phosphate Buffer Wako PureChemical Cat#16120141 Solution Corporation Dulbecco's phosphate-bufferedsaline, Thermo Fisher Cat#14040182 calcium, magnesium ScientificDulbecco's phosphate-buffered saline, no Thermo Fisher Cat#14190144calcium, no magnesium Scientific Chloroform Sigma-Aldrich Cat#288306Methanol Sigma-Aldrich Cat#1060351000 Trypsin-EDTA (0.25%) Thermo FisherCat#25200056 Scientific Collagenase, Type IV Thermo Fisher Cat#17104019Scientific Alexa Fluo 647 Phalloidin Thermo Fisher Cat#A22287 Scientific4′,6-Diamidino-2-Phenylindole, Thermo Fisher Cat#D1306 Dihydrochloride(DAPI) Scientific Anti-green fluorescent protein Alexa Fluor ThermoFisher Cat#A21311 488 conjugate Scientific Type I collagen CorningCat#354236 TrypLE Express Life Technologies Cat#12605-010 Cell recoverysolution BD Cat#354253 Collagenase I Thermo Fisher Cat#17100-017Scientific FBS Gibco Cat#10082-147 Matrigel matrix growth factor reducedCorning Cat#356231 ER-1 activation solution Emulate Inc. Cat#ER-1 ER-2activation solution Emulate Inc. Cat#ER-2 Critical commercial assays MSDU-plex Assay Meso Scale https://www.mesoscale.com Diagnostoc Rneasy MiniKit Qiagen Cat#74104 PowerUp SYBR Green Master Mix Thermo FisherCat#A25742 Scientific SuperScript IV VILO Master Mix Thermo FisherCat#11756500 Scientific Shiga Toxin 1 ELISA Abraxis Cat#542000 Depositeddata RNA-seq data Sequence Read Archive (accession: PRJNA497914])Oligonucleotides Primer: fliC 1259 Forward: (Morgan et al., 2014)GTGATGCTGCGAAGTCTTA Primer for fliC 1355 Reverse: (Morgan et al., 2014)ACAGAGCCGTTATCCTTGT Primer for rpoA: Forward: (Yin et al., 2011)TCAGGTTGAGCAGGATTTCC Primer for rpoA Reverse: (Yin et al., 2011)TGACCCTTGAGCCTTTAGAG Primer for arcA Forward: (Jandu et al., 2009)GAAGACGAGTTGGTAACACG Primer for arcA Reverse: (Jandu et al., 2009)CTTCCAGATCACCGCAGAAGC Primer for Keio Flic knockout Forward: This studyGTTCCGTTTGCCAGCCATTT Primer for Keio Flic knockout Reverse: This studyTCAGGTTGCTGCCGATGG Recombinant DNA pGEN-GFP(LVA) CbR plasmid (Wiles etal., 2009) F primer FliC 2 GTTCCGTTTGCCA GCCATTT R promer FliC 2TCAGGTTGCTGCC GATGG fliC-lux s28 flagellin gene Mobley Lab promoterfusion (AmpR) Software and algorithms Fiji (Schindelin et al.,https://fiji.sc/ 2012) StackReg plugin (Thévenaz et al.,https://imagej.net/StackReg 1998) TrackMate plugin (Tinevez et al.,2017) https://imagej.net/TrackMate R language and environment forstatistical https://www.r- computing project.org/ Linear Models forMicroarray Data (limma) (Ritchie et al., 2015) 10.18129/B9.bioc.limmapackage R metabolomics package (Bowne J B, 2014) https://cran.r-project.org/web/packages/ metabolomics/index.html DEseq package (Love etal., 2014) 10.18129/B9.bioc.DESeq ClusterProfiler (Yu et al., 2012)10.18129/B9.bioc.cluster- Profiler IMARIS Bitplane

Each of these embodiments and obvious variations thereof is contemplatedas falling within the spirit and scope of the claimed invention, whichis set forth in the following claims. Moreover, the present conceptsexpressly include any and all combinations and sub-combinations of thepreceding elements and aspects.

1-147. (canceled)
 148. A method, comprising: a) providing a microfluidicdevice, a source of microbes comprising living obligate anaerobes andliving parenchyma cells; and b) culturing the obligate anaerobes and theparenchyma cells in the microfluidic device such that at least a portionof the obligate anaerobes and at least a portion of the parenchyma cellsare in direct contact.
 149. The method of claim 148, wherein the livingobligate anaerobes are from the surface or contents of a body, orificeor cavity.
 150. The method of claim 148, wherein the parenchyma cellsare human intestinal epithelial cells.
 151. The method of claim 148,wherein, after the culturing, unknown microbes are identified.
 152. Themethod of claim 148, wherein the culturing comprises flowing media at aflow rate.
 153. The method of claim 148, wherein the microfluidic devicecomprises a second microchannel positioned below a first microchanneland separated from the first microchannel by a membrane.
 154. The methodof claim 153, wherein oxygenated medium flows through the secondmicrochannel from external oxygenated medium reservoirs.
 155. The methodof claim 154, wherein parenchyma cells in the first microchannel getoxygen from the second microchannel.
 156. The method of claim 148,wherein the culturing takes place for at least 2 days.
 157. The methodof claim 148, wherein the culturing takes place for at least 3 days.158. The method of claim 148, wherein the culturing takes place for atleast 5 days.
 159. The method of claim 157, wherein cultured microbiomecomprises both anaerobic bacteria and aerobic bacteria.
 160. The methodof claim 157, wherein the cultured microbiome comprises at least 2anaerobic species found in the fecal sample.
 161. The method of claim157, wherein the cultured microbiome comprises microbes from at least 2genera found in the fecal sample.